IL3Ralpha Antibody Conjugates and Uses Thereof

ABSTRACT

The present invention provides antibodies that bind to the IL-3 receptor alpha subunit alpha (Il3Rα) chain, and compositions comprising such antibodies. The present invention provides methods for inhibiting or reducing an IL3Rα-expressing cell population, the methods comprising contacting a population of IL3Rα-expressing cells (e.g., cancer cells and/or cancer stem cells) with an antibody that binds to IL3Rα. The present invention also provides antibody conjugates comprising an antibody that binds to an IL3Rα chain linked to a cytotoxic agent or anticellular agent and compositions comprising such conjugates. The present invention also provides methods for preventing, treating and/or managing a disorder associated with IL3Rα-expressing cells (e.g., a hematological cancer), the methods comprising administering to a subject in need thereof an antibody that binds to IL3Rα.

This application is a continuation of U.S. patent application Ser. No.15/834,211, filed on Dec. 7, 2017, which is a continuation of U.S.patent application Ser. No. 15/342,668, filed on Nov. 3, 2016 (now U.S.Pat. No. 9,873,743), which is a continuation of U.S. patent applicationSer. No. 14/632,178, filed on Feb. 26, 2015 (now U.S. Pat. No.9,518,119), which is a continuation of U.S. patent application Ser. No.13/439,453, filed on Apr. 4, 2012 (now U.S. Pat. No. 8,992,910), whichis a continuation of U.S. patent application Ser. No. 12/082,940, filedon Apr. 14, 2008 (now U.S. Pat. No. 8,163,279), which claims the benefitof U.S. Provisional Application No. 60/923,499 filed on Apr. 13, 2007,the disclosure of each of which is incorporated herein by reference inits entirety.

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2019-04-24_01214-0006-05US_Sequence_Listing.txt” created on Apr. 24,2019, which is 19,433 bytes in size. The information in the electronicformat of the sequence listing is incorporated herein by reference inits entirety.

1. FIELD OF THE INVENTION

The present invention provides methods for inhibiting or reducing anIL-3 receptor alpha subunit alpha (IL3Rα)-expressing cell population,the methods comprising contacting a population of IL3Rα-expressing cells(e.g., cancer cells and/or cancer stem cells) with an antibody thatbinds to IL3Rα. The present invention also provides methods forpreventing, treating and/or managing a disorder associated withIL3Rα-expressing cells (e.g., a hematological cancer), the methodscomprising administering to a subject in need thereof an antibody thatbinds to IL3Rα.

2. BACKGROUND OF THE INVENTION 2.1 Cancer Therapy

Cancer is one of the most significant health conditions. The AmericanCancer Society's Cancer Facts and Figures, 2003, predicts over 1.3million Americans will receive a cancer diagnosis this year. In theUnited States, cancer is second only to heart disease in mortalityaccounting for one of four deaths. In 2002, the National Institutes ofHealth estimated total costs of cancer totaled $171.6 billion, with $61billion in direct expenditures. The incidence of cancer is widelyexpected to increase as the US population ages, further augmenting theimpact of this condition. The current treatment regimens for cancerestablished in the 1970s and 1980s, have not changed dramatically. Thesetreatments, which include chemotherapy, radiation and other modalitiesincluding newer targeted therapies, have shown limited overall survivalbenefit when utilized in most advanced stage common cancers since, amongother things, these therapies primarily target tumor bulk.

More specifically, conventional cancer diagnosis and therapies to datehave attempted to selectively detect and eradicate neoplastic cells thatare largely fast-growing (i.e., cells that form the tumor bulk).Standard oncology regimens have often been largely designed toadminister the highest dose of irradiation or a chemotherapeutic agentwithout undue toxicity, i.e., often referred to as the “maximumtolerated dose” (MTD) or “no observed adverse effect level” (NOAEL).Many conventional cancer chemotherapies (e.g., alkylating agents such ascyclophosphamide, antimetabolites such as 5-Fluorouracil, and plantalkaloids such as vincristine) and conventional irradiation therapiesexert their toxic effects on cancer cells largely by interfering withcellular mechanisms involved in cell growth and DNA replication.Chemotherapy protocols also often involve administration of acombination of chemotherapeutic agents in an attempt to increase theefficacy of treatment. Despite the availability of a large variety ofchemotherapeutic agents, these therapies have many drawbacks (see, e.g.,Stockdale, 1998, “Principles Of Cancer Patient Management” in ScientificAmerican Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect.X). For example, chemotherapeutic agents are notoriously toxic due tonon-specific side effects on fast-growing cells whether normal ormalignant; e.g. chemotherapeutic agents cause significant, and oftendangerous, side effects, including bone marrow depression,immunosuppression, and gastrointestinal distress, etc.

Other types of traditional cancer therapies include surgery, hormonaltherapy, immunotherapy, anti-angiogenesis therapy, targeted therapy(e.g., therapy directed to a cancer target such as Gleevec® and othertyrosine kinase inhibitors, Velcade®, Sutent®, et al.), and radiationtreatment to eradicate neoplastic cells in a patient (see, e.g.,Stockdale, 1998, “Principles of Cancer Patient Management,” inScientific American: Medicine, vol. 3, Rubenstein and Federman, eds.,ch. 12, sect. IV). All of these approaches can pose significantdrawbacks for the patient including a lack of efficacy (in terms oflong-term outcome (e.g. due to failure to target cancer stem cells) andtoxicity (e.g. due to non-specific effects on normal tissues)).Accordingly, new therapies for improving the long-term prospect ofcancer patients are needed.

2.2 Cancer Stem Cells

Cancer stem cells comprise a unique subpopulation (often 0.1-10% or so)of a tumor that, relative to the remaining 90% or so of the tumor (i.e.,the tumor bulk), are more tumorigenic, relatively more slow-growing orquiescent, and often relatively more chemoresistant than the tumor bulk.Given that conventional therapies and regimens have, in large part, beendesigned to attack rapidly proliferating cells (i.e. those cancer cellsthat comprise the tumor bulk), cancer stem cells which are oftenslow-growing may be relatively more resistant than faster growing tumorbulk to conventional therapies and regimens. Cancer stem cells canexpress other features which make them relatively chemoresistant such asmulti-drug resistance and anti-apoptotic pathways. The aforementionedwould constitute a key reason for the failure of standard oncologytreatment regimens to ensure long-term benefit in most patients withadvanced stage cancers—i.e. the failure to adequately target anderadicate cancer stem cells. In some instances, a cancer stem cell(s) isthe founder cell of a tumor (i.e., it is the progenitor of the cancercells that comprise the tumor bulk).

Cancer stem cells have been identified in a large variety of cancertypes. For instance, Bonnet et al., using flow cytometry were able toisolate the leukemia cells bearing the specific phenotype CD34+CD38−,and subsequently demonstrate that it is these cells (comprising <1% of agiven leukemia), unlike the remaining 99+% of the leukemia bulk, thatare able to recapitulate the leukemia from whenst it was derived whentransferred into immunodeficient mice. See, e.g., “Human acute myeloidleukemia is organized as a hierarchy that originates from a primitivehematopoietic cell,” Nat. Med. 3:730-737 (1997). That is, these cancerstem cells were found as <1 in 10,000 leukemia cells yet this lowfrequency population was able to initiate and serially transfer a humanleukemia into severe combined immunodeficiency/non-obese diabetic(NOD/SCID) mice with the same histologic phenotype as in the originaltumor.

Cox et al. identified small subfractions of human acute lymphoblasticleukemia (ALL) cells which had the phenotypes CD34⁺/CD10⁻ andCD34⁺/CD19⁻, and were capable of engrafting ALL tumors inimmunocompromised mice—i.e. the cancer stem cells. In contrast, noengraftment of the mice was observed using the ALL bulk, despite, insome cases, injecting 10-fold more cells. See Cox et al.,“Characterization of acute lymphoblastic leukemia progenitor cells,”Blood 104(19): 2919-2925 (2004).

Multiple myeloma was found to contain small subpopulations of cells thatwere CD138− and, relative to the large bulk population of CD138+ myelomacells, had greater clonogenic and tumorigenic potential. See Matsui etal., “Characterization of clonogenic multiple myeloma cells,” Blood103(6): 2332. The authors concluded that the CD138− subpopulation ofmultiple myeloma was the cancer stem cell population.

Kondo et al. isolated a small population of cells from a C6-glioma cellline, which was identified as the cancer stem cell population by virtueof its ability to self-renew and recapitulate gliomas inimmunocompromised mice. See Kondo et al., “Persistence of a smallpopulation of cancer stem-like cells in the C6 glioma cell line,” Proc.Natl. Acad. Sci. USA 101:781-786 (2004). In this study, Kondo et al.determined that cancer cell lines contain a population of cancer stemcells that confer the ability of the line to engraft immunodeficientmice.

Breast cancers were shown to contain a small population of cells withstem cell characteristics (bearing surface markersCD44+CD24^(low lin-)). See Al-Hajj et al., “Prospective identificationof tumorigenic breast cancer cells,” Proc. Natl. Acad. Sci. USA100:3983-3988 (2003). As few as 200 of these cells, corresponding to1-10% of the total tumor cell population, are able to form tumors inNOD/SCID mice. In contrast, implantation of 20,000 cells that lackedthis phenotype (i.e. the tumor bulk) was unable to re-grow the tumor.

A subpopulation of cells derived from human prostate tumors was found toself-renew and to recapitulate the phenotype of the prostate tumor fromwhich they were derived thereby constituting the prostate cancer stemcell population. See Collins et al., “Prospective Identification ofTumorigenic Prostate Cancer Stem Cells,” Cancer Res 65(23):10946-10951(2005).

Fang et al. isolated a subpopulation of cells from melanoma with cancerstem cell properties. In particular, this subpopulation of cells coulddifferentiate and self-renew. In culture, the subpopulation formedspheres whereas the more differentiated cell fraction from the lesionswere more adherent. Moreover, the subpopulation containing sphere-likecells were more tumorigenic than the adherent cells when grafted intomice. See Fang et al., “A Tumorigenic Subpopulation with Stem CellProperties in Melanomas,” Cancer Res 65(20): 9328-9337 (2005).

Singh et al. identified brain tumor stem cells. When isolated andtransplanted into nude mice, the CD133+ cancer stem cells, unlike theCD133− tumor bulk cells, form tumors that can then be seriallytransplanted. See Singh et al., “Identification of human brain tumorinitiating cells,” Nature 432:396-401 (2004); Singh et al., “Cancer stemcells in nervous system tumors,” Oncogene 23:7267-7273 (2004); Singh etal., “Identification of a cancer stem cell in human brain tumors,”Cancer Res. 63:5821-5828 (2003).

Since conventional cancer therapies target rapidly proliferating cells(i.e., cells that form the tumor bulk) these treatments are believed tobe relatively ineffective at targeting and impairing cancer stem cells.In fact, cancer stem cells, including leukemia stem cells, have indeedbeen shown to be relatively resistant to conventional chemotherapeutictherapies (e.g. Ara-C, daunorubicin) as well as newer targeted therapies(e.g. Gleevec®, Velcade®). Examples of cancer stem cells from varioustumors that are resistant to chemotherapy, and the mechanism by whichthey are resistant, are described in Table 1 below.

TABLE 1 CSC Type Resistance Mechanism Reference AML Ara-C QuiescenceGuzman. Blood ′01 AML Daunorubicin Drug Efflux, Anti- Costello. CancerRes apoptosis ′00 AML Daunorubicin, Drug Efflux Wulf. Blood ′01mitoxantrone AML Quiescence Guan. Blood ′03 AML, Anti-apoptosis Suarez.Clin Cancer MDS Res ′04 CML Quiescence Holyoake. Blood ′99 CML Gleevec ®Quiescence Graham. Blood ′02 Myeloma Velcade ® Matsui. ASH 04

For example, leukemic stem cells are relatively slow-growing orquiescent, express multi-drug resistance genes, and utilize otheranti-apoptotic mechanisms—features which contribute to theirchemoresistance. See Jordan et al., “Targeting the most critical cells:approaching leukemia therapy as a problem in stem cell biology,” Nat.Clin. Pract. Oncol. 2: 224-225 (2005). Further, cancer stem cells byvirtue of their chemoresistance may contribute to treatment failure, andmay also persist in a patient after clinical remission and theseremaining cancer stem cells may therefore contribute to relapse at alater date. See Behbood et al., “Will cancer stem cells provide newtherapeutic targets?” Carcinogenesis 26(4): 703-711 (2004). Therefore,targeting cancer stem cells is expected to provide for improvedlong-term outcomes for cancer patients. Accordingly, new therapeuticagents and/or regimens designed to target cancer stem cells are neededto reach this goal.

2.3 Acute Myeloid Leukemia

Approximately forty thousand patients per year develop acute myeloidleukemia (AML) in the U.S., Canada, and Europe. See, e.g., Jamal et al.,Cancer Statisitics 56:106-130 (2006). AML is the most common leukemia inadults and the second most common leukemia in children. The prolongedhospitalizations associated with treatment and complications represent asignificant share of health care costs in these regions. Further, evenwith combination induction and consolidation chemotherapy, most patientsultimately relapse and die from their disease or complications oftreatment. See, e.g., Brune et al., “Improved leukemia-free survivalafter post-consolidation immunotherapy with histamine dihydrochlorideand interleukin-2 in acute myeloid leukemia: results of a randomizedphase III trial,” Blood 108(1):88-96 (2006). Novel therapies areurgently needed. Selective targeting of AML cells stem cells may providea safe and more effective therapy.

2.4 Myelodysplastic Syndrome

There are approximately 20,000 new cases of myelodysplastic syndrome(MDS) each year in the U.S. Patients with myelodysplastic syndromestypically have low blood cell counts in at least one or more of redblood cells, white blood cells, and platelets. Upon examination, thebone marrow usually is found to be dysplastic or hyperplastic, meaningthere are too many poorly functioning blood stem cells in the marrow. Asmall percentage of MDS patients have hypoplastic bone marrow, meaningthere are too few blood stem cells in the marrow, which make the diseaselook similar to aplastic anemia. Nearly half of people with MDS have nosymptoms at time of diagnosis. When signs and symptoms do occur they caninclude anemia, weakness, fatigue, headache, bruising, increasedbleeding, rash, fevers, mouth sores and lingering illness. MDS occurs atan increasing frequency in older people, but it can occur in childrentoo. In less than a third of patients, MDS progresses over time tobecome acute leukemia. The average age of diagnosis is 70 years old.Treatments for MDS may vary considerably, depending on the type of MDS,the history of the patient, and the age and ability to tolerate certaintreatment regimens. Treatment options include supportive care,chemotherapy-related agents, and stem cell transplantation (which istypically used only in patients under 50). However, the remission ratefor existing treatments in relatively low, and new therapies are needed.

2.5 Interleukin-3 and the IL-3 Receptor Alpha Subunit

Interleukin-3 (IL-3) is a cytokine that supports the proliferation anddifferentiation of multipotential and committed myeloid and lymphoidprogenitors, but is not required for growth and differentiation ofnormal stem cells and mature myeloid cells. See, e.g., Nitsche et al.,“Interleukin-3 promotes proliferation and differentiation of humanhematopoietic stem cells but reduces their repopulation potential inNOD/SCID mice,” Stem Cells 21:236-244 (2003).

Human IL-3 mediates its effects by binding to the human IL-3 receptor,which is a heterodimer consisting of an IL-3—binding alpha subunit(IL3Rα, CD123) and a common beta subunit (IL3Rβ_(c)). The alpha subunitof IL3R is essential for ligand binding and specificity. The α subunitis essential for ligand binding and confers specificity on the receptor.The β subunit is also shared by the granulocyte macrophage-colonystimulating factor (GM-CSF) and IL-5 receptors, and is required for highaffinity ligand binding and signal transduction.

The alpha subunit of the human IL-3 receptor is strongly expressed in45%-95% of AML, 40%-100% of B-cell lineage acute lymphocytic leukemia(B-ALL), and 85% of hairy cell leukemia (HCL). See, e.g., Graf et al.,“Expression and prognostic value of hemopoietic cytokine receptors inacute myeloid leukemia (AML): implications for future therapeuticalstrategies,” Eur. J. Haematol. 72:89-106 (2004); Jordan et al., “TheIL-3 receptor alpha subunit alpha chain is a unique marker for humanacute myelogenous leukemia stem cells,” Leukemia 14:1777-1784 (2000);Munoz et al., “IL-3 receptor alpha subunit alpha chain (CD123) is widelyexpressed in hematologic malignancies,” Haematologica 86:1261-1269(2001); Testa et al., “Elevated expression of IL-3R alpha in acutemyelogenous leukemia is associated with enhanced blast proliferation,increased cellularity, and poor prognosis,” Blood 100:2980-2988 (2002).The common β subunit of the IL-3 receptor is shared by the IL-3,granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5receptors. See, e.g., Testa et al., “IL-3 receptor alpha subunit inacute leukemia,” Leukemia 18:219-226 (2004). The expression of IL3Rα iselevated in the primitive LSC population (CD34+/CD38−) in AML, chronicmyelogenous leukemia (CML), and some other myeloid malignancies, but isnot detectable in normal hematopoietic cells and progenitors. See, e.g.,Florian et al., “Detection of molecular targets on the surface ofCD34+/CD38− stem cells in various myeloid malignancies,” Leuk. Lymphoma47:207-222 (2006); Jordan et al., 2000, above; Hogge et al., “VariantDiphtheria Toxin-Interleukin-3 Conjugates with Increased ReceptorAffinity Have Enhanced Cytotoxicity against Acute Myeloid LeukemiaProgenitors,” Clin. Cancer Res. 12:1284-1291 (2004). Thus, IL3Rα appearsto be an excellent target for the therapy of leukemias.

3. SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprising anantibody that binds to the IL3Rα chain, and a pharmaceuticallyacceptable carrier or excipient. In one aspect, the invention providespharmaceutical compositions comprising an antibody that binds to theIL3Rα chain in an amount effective to reduce cancer stem cells and/orcancer cells in an animal with or animal model for myeloid leukemia oranother cancer associated with IL3Rα-expressing cells by about 25%,about 30%, 35%, about 40%, about 45%, about 50%, about 65%, about 75% ormore relative to a negative control. In a specific embodiment, theantibody incorporated into a pharmaceutical composition has adissociation constant (K_(d)) of less than 4 nM on cells expressing theIL3Rα chain (e.g., erytholeukemic cells, myeloid cells or myeloblasticcells). In another embodiment, the antibody incorporated into apharmaceutical composition comprises a variable heavy (VH) domain havingthe amino acid sequence of SEQ ID NO:2, 12, 22, or 32, and/or a variablelight (VL) domain having the amino acid sequence of SEQ ID NO: 7, 17, 27or 37. In another embodiment, the antibody incorporated into apharmaceutical composition comprises one or more of the VHcomplementarity determining regions (CDRs) shown in FIGS. 1B, 2B, 3Band/or 4B and/or a VL domain comprising one or more of the VL CDRs shownin FIGS. 1D, 2D, 3D and/or 4D. In another embodiment, the antibodyincorporated into a pharmaceutical composition competes with an antibodycomprising a VH domain having the amino acid sequence of SEQ ID NO:2,12, 22, or 32 and a VL domain having the amino acid sequence of SEQ IDNO: 7, 17, 27 or 37 for binding to the IL3Rα chain. In some embodiments,a pharmaceutical composition of the invention comprises an agent inaddition to an antibody of the invention.

The present invention also provides methods for inhibiting theproliferation or reducing an IL3Rα-expressing cell population, themethods comprising contacting a population of cells comprisingIL3Rα-expressing cells with an antibody that binds to the IL3 Rα chain.In a specific embodiment, the present invention provides methods forinhibiting the proliferation or reducing the population of cancer stemcells expressing IL3Rα, the methods comprising contacting theIL3Rα-expressing cancer stem cell population with an antibody that bindsto the IL3Rα chain. In another embodiment, the present inventionprovides methods for inhibiting the proliferation or reducing thepopulation of cancer cells expressing IL3Rα, the methods comprisingcontacting the IL3Rα-expressing cancer cell population with an antibodythat binds to the IL3Rα chain. In yet another embodiment, the presentinvention provides methods for inhibiting the proliferation or reducingthe population cancer cells and cancer stem cells expressing IL3Rα, themethods comprising contacting a population of cells comprisingIL3Rα-expressing cancer cells and/or IL3Rα-expressing cancer stem cellswith an antibody that binds to the IL3Rα chain. In certain embodiments,the antibody reduces a cancer stem cell population and/or a cancer cellpopulation by at least 25%, at least 30%, at least 40%, at least 50%, atleast 65%, at least 75%, or at least 85% in an animal with or animalmodel for myeloid leukemia or another cancer associated withIL3Rα-expressing cells relative to a negative control.

The present invention also provides methods for preventing, treatingand/or managing a disorder associated with IL3Rα-expressing cells (e.g.,a hematologic cancer), the methods comprising administering to a subjectin need thereof an antibody that binds to the IL3 Rα chain. In aspecific embodiment, the present invention provides a method forpreventing, treating and/or managing a disorder associated withIL3Rα-expressing cells (e.g., a hematologic cancer), the methodcomprising administering to a human subject in need thereof an effectiveamount of an antibody that binds to the IL3 Rα chain, and in someembodiments, an effective amount of another therapy. Non-limitingexamples of disorders associated with IL3Rα-expressing cells includeautoimmune disorders (such as lupus), inflammatory disorders (such asallergies and asthma) and cancers (such as hematological cancers). Insome embodiments, the disorder associated with IL3Rα-expressing cells isnot myeloid leukemia.

The present invention provides methods for preventing relapse of cancerassociated with IL3Rα-expressing cells, the methods comprisingadministering to a subject in need thereof an antibody that binds to theIL3 Rα chain. In a specific embodiment, the present invention providesmethods for preventing relapse of cancer associated withIL3Rα-expressing cells, the methods comprising administering to a humansubject in need thereof an effective amount of an antibody that binds tothe IL3 Rα chain, and in some embodiments, an effective amount ofanother therapy. In some embodiments, the cancer associated withIL3Rα-expressing cells is not myeloid leukemia.

The present invention provides methods for preventing, treating and/ormanaging cancer associated with IL3Rα-expressing cells (e.g., ahematologic cancer), the method comprising administering to a humansubject in need thereof an antibody that binds to the IL3 Rα chain in anamount effective to reduce the quantity, number, amount or percentage ofcancer stem cells and/or cancer cells by at least 25%, at least 30%, atleast 40%, at least 50%, at least 65%, at least 75%, or at least 85% inan animal with or animal model for myeloid leukemia or another cancerassociated with IL3Rα-expressing cells relative to a negative control.

The present invention provides antibody conjugates comprising anantibody that binds to the alpha chain of the IL-3 receptor alphasubunit (IL3Rα, CD123) linked to a cytotoxic agent or other moiety, andcompositions comprising such conjugates and uses of such conjugates,including the prevention, management and/or treatment of a disorderassociated with IL3Rα-expressing cells. In a specific embodiment, theantibody conjugate is an immunotoxin. In one aspect, the presentinvention provides an antibody conjugate comprising an antibody thatbinds to the IL3Rα chain linked to a cytotoxic agent, wherein theantibody conjugate has an IC50 of less than 40 ng/ml on cells expressingthe IL3Rα chain. In a specific embodiment, the antibody has adissociation constant (K_(d)) of less than 4 nM on cells expressing theIL3Rα chain (e.g., erytholeukemic cells, myeloid cells or myeloblasticcells).

In another aspect, the present invention provides an antibody conjugatecomprising an antibody that binds to the IL3Rα chain linked to acytotoxic agent, wherein the antibody comprises a variable heavy (VH)domain having the amino acid sequence of SEQ ID NO:2, 12, 22, or 32,and/or a variable light (VL) domain having the amino acid sequence ofSEQ ID NO: 7, 17, 27 or 37. In one embodiment, the antibody comprises avariable heavy (VH) domain having the amino acid sequence of SEQ ID NO:2(FIG. 1B), and/or a variable light (VL) domain having the amino acidsequence of SEQ ID NO: 7 (FIG. 1D). In another embodiment, the antibodycomprises a variable heavy (VH) domain having the amino acid sequence ofSEQ ID NO: 12 (FIG. 2B), and/or a variable light (VL) domain having theamino acid sequence of SEQ ID NO: 17 (FIG. 2D). In yet anotherembodiment, the antibody comprises a variable heavy (VH) domain havingthe amino acid sequence of SEQ ID NO: 22 (FIG. 3B), and/or a variablelight (VL) domain having the amino acid sequence of SEQ ID NO: 27 (FIG.3D). In yet another embodiment, the antibody comprises a variable heavy(VH) domain having the amino acid sequence of SEQ ID NO: 32 (FIG. 4B),and/or a variable light (VL) domain having the amino acid sequence ofSEQ ID NO: 37 (FIG. 4D).

In another aspect, the present invention provides an antibody conjugatecomprising an antibody that binds to the ILR3α chain linked to acytotoxic agent, wherein the antibody comprises one or more of the VHcomplementarity determining regions (CDRs) shown in FIGS. 1B, 2B, 3Band/or 4B and/or a VL domain comprising one or more of the VL CDRs shownin FIGS. 1D, 2D, 3D and/or 4D. In one embodiment, the antibody comprisesa VH domain comprising one or more of the VH CDRs shown in FIG. 1B (SEQID NO: 3, 4, or 5) and/or a VL domain comprising one or more of the VLCDRs shown in FIG. 1D (SEQ ID NO: 8, 9, or 10). In another embodiment,the antibody comprises a VH domain comprising one or more of the VH CDRsshown in FIG. 2B (SEQ ID NO: 13, 14, or 15) and/or a VL domaincomprising one or more of the VL CDRs shown in FIG. 2D (SEQ ID NO: 18,19, or 20). In another embodiment, the antibody comprises a VH domaincomprising one or more of the VH CDRs shown in FIG. 3B (SEQ ID NO: 23,24, or 25) and/or a VL domain comprising one or more of the VL CDRsshown in FIG. 3D (SEQ ID NO: 28, 29, or 30). In yet another embodiment,the antibody comprises a VH domain comprising one or more of the VH CDRsshown in FIG. 4B (SEQ ID NO: 33, 34, or 35) and/or a VL domaincomprising one or more of the VL CDRs shown in FIG. 4D (SEQ ID NO: 38,39, or 40).

In another aspect, the present invention provides an antibody conjugatecomprising an antibody that binds to the IL3Rα chain linked to acytotoxic agent, wherein the antibody competes with an antibodycomprising a VH domain having the amino acid sequence of SEQ ID NO:2,12, 22, or 32 and a VL domain having the amino acid sequence of SEQ IDNO: 7, 17, 27 or 37 for binding to the IL3Rα chain. In a specificembodiment, the present invention provides an antibody conjugatecomprising an antibody that binds to the IL3Rα chain linked to acytotoxic agent, wherein the antibody binds to the same epitope or anepitope that overlaps the epitope of an antibody comprising a VH domainhaving the amino acid sequence of SEQ ID NO:2, 12, 22, or 32 and a VLdomain having the amino acid of SEQ ID NO: 7, 17, 27 or 37. In someembodiments, the antibody has a dissociation constant (K_(d)) of lessthan 4 nM on cells expressing the IL3Rα chain (e.g., erytholeukemiccells, myeloid cells or myeloblastic cells).

In some embodiments, an antibody conjugate of the invention comprises acytotoxic agent that is non-proteinaceous. In accordance with theseembodiments, the cytotoxic agent can be chemically conjugated to theantibody, either directly or through a chemical linker. In otherembodiments, an antibody conjugate of the invention comprises acytotoxic agent that is proteinaceous. In accordance with theseembodiments, the cytotoxic agent can be covalently linked to theantibody through either a peptide bond or other chemical conjugation.Non-limiting examples of cytotoxic agents include diphtheria toxin,Pseudomonas exotoxin, ribosome inactivating proteins, ricin A,deglycosylated ricin A chain, abrin, alpha sarcin, aspergillin,restrictocin, ribonucleases, bacterial endotoxin, the lipid A moiety ofbacterial endotoxin, and cholera toxin. Other examples of cytotoxicagents include, but are not limited to, peptides derived from proteinsinvolved in apoptosis, such as Bcl-x, Bax, or Bad. In one embodiment,the cytotoxic agent is Pseudomonas exotoxin A or a fragment thereof. Ina specific embodiment, the cytotoxic agent is a fragment of Pseudomonasexotoxin A that lacks the native receptor binding domain and containsthe translocation and ADP-ribosylation domains of Pseudomonas exotoxinA. In another specific embodiment, the cytotoxic agent is a fragment ofPseudomonas exotoxin A that has been modified at its carboxyl terminusso that it has the amino acid sequence Lys-Asp-Glu-Leu (KDEL).

The present invention provides compositions comprising an antibodyconjugate of the invention, and a carrier or excipient. In oneembodiment, the present invention provides pharmaceutical compositionscomprising an antibody conjugate of the invention, and apharmaceutically acceptable carrier or excipient. In a specificembodiment, the invention provides pharmaceutical compositionscomprising an antibody conjugate of the invention in an amount effectiveto reduce the quantity, number, amount or percentage of cancer stemcells and/or cancer cells in an animal with or animal model for myeloidleukemia or another cancer by about 25%, about 30%, 35%, about 40%,about 45%, about 50%, about 65%, about 75% or more relative to anegative control. In some embodiments, the compositions of the inventioncomprise an agent in addition to the antibody conjugate.

The present invention provides methods for inhibiting the proliferationor reducing the IL3Rα-expressing cell population, the methods comprisingcontacting a population of cells comprising IL3Rα-expressing cells withan antibody conjugate of the invention. In a specific embodiment, thepresent invention provides methods for inhibiting the proliferation orreducing the population of cancer stem cells expressing IL3Rα, themethods comprising contacting the IL3Rα-expressing cancer stem cellpopulation with an antibody conjugate of the invention. In anotherembodiment, the present invention provides methods for inhibiting theproliferation or reducing the population of cancer cells expressingIL3Rα, the methods comprising contacting the IL3Rα-expressing cancercell population with an antibody conjugate of the invention. In yetanother embodiment, the present invention provides methods forinhibiting the proliferation or reducing the population cancer cells andcancer stem cells expressing IL3Rα, the methods comprising contacting apopulation of cells comprising IL3Rα-expressing cancer cells and/orIL3Rα-expressing cancer stem cells with an antibody conjugate of theinvention. In some embodiments, the antibody conjugate reduces a cancerstem cell population and/or a cancer cell population by at least 25%, atleast 30%, at least 40%, at least 50%, at least 65%, at least 75% or atleast 85% in an animal with or animal model for myeloid leukemia oranother cancer associated with IL3Rα-expressing cells relative to anegative control.

The present invention provides methods for preventing, treating and/ormanaging a disorder associated with IL3Rα-expressing cells (e.g., ahematologic cancer), the methods comprising administering to a subjectin need thereof an antibody conjugate of the invention. In a specificembodiment, the present invention provides a method for preventing,treating and/or managing a disorder associated with IL3Rα-expressingcells (e.g., a hematologic cancer), the method comprising administeringto a human subject in need thereof an effective amount of an antibodyconjugate of the invention, and in some embodiments, an effective amountof another therapy. Non-limiting examples of disorders associated withIL3Rα-expressing cells include autoimmune disorders (such as lupus),inflammatory disorders (such as allergies and asthma) and cancers (suchas hematologic cancers). In some embodiments, the disorder associatedwith IL3Rα-expressing cells is not myeloid leukemia.

The present invention provides methods for preventing relapse of cancerassociated with IL3Rα-expressing cells, the methods comprisingadministering to a subject in need thereof an antibody conjugate of theinvention. In a specific embodiment, the present invention providesmethods for preventing relapse of cancer associated withIL3Rα-expressing cells, the methods comprising administering to a humansubject in need thereof an effective amount of an antibody conjugate ofthe invention, and in some embodiments, an effective amount of anothertherapy. In some embodiments, the cancer associated withIL3Rα-expressing cells is not myeloid leukemia.

The present invention provides methods for preventing, treating and/ormanaging cancer associated with IL3Rα-expressing cells (e.g., ahematological cancer), the method comprising administering to a humansubject in need thereof an antibody conjugate of the invention in anamount effective to reduce the quantity, number, amount or percentage ofcancer stem cells and/or cancer cells by at least 25%, at least 30%, atleast 40%, at least 50%, at least 65%, at least 75%, or at least 85% inan animal with or animal model for myeloid leukemia or another cancerassociated with IL3Rα-expressing cells relative to a negative control.

The present invention provides methods for ex vivo purging of bonemarrow or peripheral blood to remove cells that express IL3Rα such thatthe purged bone marrow or peripheral blood is suitable, e.g., forautologous stem cell transplantation to restore hematopoeitic function.

3.1 Definitions

As used herein, the terms “about” or “approximately,” unless otherwiseindicated, refer to a value that is no more than 10% above or below thevalue being modified by the term.

As used herein, the term “agent” refers to any molecule, compound,and/or substance for use in the prevention, treatment, management and/ordiagnosis of a disease, including but not limited to cancer, autoimmunedisease, or allergic disease. Included in this definition is the IL3Rαantibody-toxin conjugate of the invention.

As used herein, the terms “alpha subunit of the IL-3 receptor,” “IL3Rα,”“CD123,” “IL3Rα chain” and “IL3Rα subunit” refer interchangeably to anantigenic determinant detectable on leukemia precursor cells, whichimmunobinds interleukin-3 (IL3). The human and murine amino acid andnucleic acid sequences can be found, e.g., in a public database such asGenBank. For example, the amino acid sequence of human IL3Rα can befound at Accession No. NP 002174 and the nucleotide sequence encoding ofthe human IL3Rα can be found at Accession No. NM 005191. In a specificembodiment, the IL3Rα is the human IL3Rα.

As used herein, the terms “antibody” and “antibodies” refer to moleculesthat contain an antigen binding site, e.g., immunoglobulins, orfragments of immunoglobulins that have the ability to bind to aparticular antigen. Immunoglobulin molecules can be of any type (e.g.,IgG, IgE, IgM, IgD, IgA, IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,IgA1, IgA2), or subclass. Antibodies include, but are not limited to,monoclonal antibodies, polyclonal antibodies, multispecific antibodies,human antibodies, humanized antibodies, camelized antibodies, chimericantibodies, single domain antibodies, single chain antibodies, Fabfragments, F(ab′) fragments, Fv fragments, single chain Fvs (scFv),disulfide-linked Fvs (dsFv), Fd, VH, VL, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. As usedherein, unless specified otherwise, the term: 26292, the 32703, the32701 or the 32716 antibody refers to the monoclonal antibody or thescFv antibody. The terms “IL3Rα antibody,” IL3Rα antibodies,”“antibodies of the invention,” “anti-IL3Rα antibody” and “anti-IL3Rαantibodies” are used interchangeably to refer to antibodies describedherein.

As used herein, the terms “antibody conjugate(s)” and “antibody fragmentconjugate(s)” refer to a conjugate(s) of an antibody or antibodyfragment that is prepared by way of a synthetic chemical reaction(s) oras a recombinant fusion protein(s).

As used herein, the term “amount,” as used in the context of the amountof a particular cell population or cells, refers to the frequency,quantity, percentage, relative amount, or number of the particular cellpopulation or cells.

As used herein, the term “bind” or “bind(s)” refers to any interaction,whether direct or indirect, that affects the specified receptor orreceptor subunit.

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. Non-limitingexamples include those cancers described in Section 5.8.3, infra. Theterm “cancer” encompasses a disease involving both pre-malignant andmalignant cancer cells. In some embodiments, cancer refers to alocalized overgrowth of cells that has not spread to other parts of asubject, i.e., a benign tumor. In other embodiments, cancer refers to amalignant tumor, which has invaded and destroyed neighbouring bodystructures and spread to distant sites. In yet other embodiments, thecancer is associated with a specific cancer antigen.

As used herein, the term “cancer cells” refers to cells that acquire acharacteristic set of functional capabilities during their development,including the ability to evade apoptosis, self-sufficiency in growthsignals, insensitivity to anti-growth signals, tissueinvasion/metastasis, significant growth potential, and/or sustainedangiogenesis. The term “cancer cell” is meant to encompass bothpre-malignant and malignant cancer cells.

As used herein, the term “cancer stem cell(s)” refers to a cell that canbe a progenitor of a highly proliferative cancer cell. A cancer stemcell has the ability to re-grow a tumor as demonstrated by its abilityto form tumors in immunocompromised mice, and typically to form tumorsupon subsequent serial transplantation in immunocompromised mice. Cancerstem cells are also typically slow-growing relative to the bulk of atumor; that is, cancer stem cells are generally quiescent. In certainembodiments, but not all, the cancer stem cell may representapproximately 0.1 to 10% of a tumor.

As used herein, the term “cytotoxin” or the phrase “cytotoxic agent”refers to a compound that exhibits an adverse effect on cell growth orviability. Included in this definition are compounds that kill cells orwhich impair them with respect to growth, longevity, or proliferativeactivity.

As used herein, the term “derivative” in the context of proteinaceousagent (e.g., proteins, polypeptides, peptides, and antibodies) refers toa proteinaceous agent that comprises an amino acid sequence which hasbeen altered by the introduction of amino acid residue substitutions,deletions, and/or additions. The term “derivative” as used herein alsorefers to a proteinaceous agent which has been modified, i.e., by thecovalent attachment of any type of molecule to the proteinaceous agent.For example, but not by way of limitation, an antibody may be modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. A derivative of a proteinaceous agent may be produced by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis in the presence of tunicamycin, etc.Further, a derivative of a proteinaceous agent may contain one or morenon-classical amino acids. A derivative of a proteinaceous agentpossesses a similar or identical function as the proteinaceous agentfrom which it was derived. The term “derivative” in the context of aproteinaceous agent also refers to a proteinaceous agent that possessesa similar or identical function as a second proteinaceous agent (i.e.,the proteinaceaous agent from which the derivative was derived) but doesnot necessarily comprise a similar or identical amino acid sequence ofthe second proteinaceous agent, or possess a similar or identicalstructure of the second proteinaceous agent. A proteinaceous agent thathas a similar amino acid sequence refers to a second proteinaceous agentthat satisfies at least one of the following: (a) a proteinaceous agenthaving an amino acid sequence that is at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% identical to the amino acidsequence of a second proteinaceous agent; (b) a proteinaceous agentencoded by a nucleotide sequence that hybridizes under stringentconditions to a nucleotide sequence encoding a second proteinaceousagent of at least 5 contiguous amino acid residues, at least 10contiguous amino acid residues, at least 15 contiguous amino acidresidues, at least 20 contiguous amino acid residues, at least 25contiguous amino acid residues, at least 40 contiguous amino acidresidues, at least 50 contiguous amino acid residues, at least 60contiguous amino residues, at least 70 contiguous amino acid residues,at least 80 contiguous amino acid residues, at least 90 contiguous aminoacid residues, at least 100 contiguous amino acid residues, at least 125contiguous amino acid residues, or at least 150 contiguous amino acidresidues; and (c) a proteinaceous agent encoded by a nucleotide sequencethat is at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least99% identical to the nucleotide sequence encoding a second proteinaceousagent. A proteinaceous agent with similar structure to a secondproteinaceous agent refers to a proteinaceous agent that has a similarsecondary, tertiary or quaternary structure to the second proteinaceousagent. The structure of a proteinaceous agent can be determined bymethods known to those skilled in the art, including but not limited to,peptide sequencing, X-ray crystallography, nuclear magnetic resonance,circular dichroism, and crystallographic electron microscopy. In aspecific embodiment, a derivative is a functionally active derivative.

As used herein, the phrase “detectable agents” refers to any molecule,compound and/or substance that is detectable by any methodologyavailable to one of skill in the art. Non-limiting examples ofdetectable agents include dyes, gas, metals, or radioisotopes.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a pathological condition in a subject.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to result in the prevention of thedevelopment, recurrence, or onset of cancer and one or more symptomsthereof, to enhance or improve the prophylactic effect(s) of anothertherapy, reduce the severity, the duration of cancer, ameliorate one ormore symptoms of cancer, prevent the advancement of cancer, causeregression of cancer, and/or enhance or improve the therapeuticeffect(s) of another therapy. In an embodiment of the invention, theamount of a therapy is effective to achieve one, two, three or more ofthe following results following the administration of one, two, three ormore therapies: (1) a stabilization, reduction or elimination of thecancer stem cell population; (2) a stabilization, reduction orelimination in the cancer cell population; (3) a stabilization orreduction in the growth of a tumor or neoplasm; (4) an impairment in theformation of a tumor; (5) eradication, removal, or control of primary,regional and/or metastatic cancer; (6) a reduction in mortality; (7) anincrease in disease-free, relapse-free, progression-free, and/or overallsurvival, duration, or rate; (8) an increase in the response rate, thedurability of response, or number of patients who respond or are inremission; (9) a decrease in hospitalization rate; (10) a decrease inhospitalization lengths; (11) the size of the tumor is maintained anddoes not increase or increases by less than 10%, preferably less than5%, preferably less than 4%, preferably less than 2%; (12) an increasein the number of patients in remission; (13) an increase in the lengthor duration of remission; (14) a decrease in the recurrence rate ofcancer; (15) an increase in the time to recurrence of cancer; and (16)an amelioration of cancer-related symptoms and/or quality of life.

As used herein, the phrase “elderly human” refers to a human 65 yearsold or older, preferably 70 years old or older.

As used herein, the term “fragment,” in the context of a proteinaceousagent (e.g., a protein or polypeptide), refers to a proteinaceous agentthat is composed of a minimum of 5 amino acids, and is 8 or more aminoacids, 10 or more amino acids, 15 or more amino acids, 20 or more aminoacids, 25 or more amino acids, 50 or more amino acids, 75 or more aminoacids, 100 or more amino acids, 150 or more amino acids, 200 or moreamino acids, 300 or more amino acids, 10 to 150 amino acids, 10 to 200amino acids, 10 to 250 amino acids, 10 to 300 amino acids, 50 to 100amino acids, 50 to 150 amino acids, 50 to 200 amino acids, 50 to 250amino acids, 50 to 300 amino acids, 50 to 350 amino acids, 50 to 400amino acids, 50 to 450 amino acids, 50 to 500 amino acids, 50 to 550amino acids, 50 to 600 amino acids, or 50 to 650 amino acids of a secondproteinaceous agent.

As used herein, the term “functionally active derivative” in the contextof a proteinaceous agent is a derivative of a proteinaceous agent thatretains at least one, multiple, or all functions of the polypeptide orprotein from which the derivative is derived. In a specific embodiment,a functionally active derivative of Pseudomonas exotoxin A retains theADP-ribosylation and translocation activities that render it cytotoxic,but lacks the receptor binding function of its parent.

As used herein, the term “functionally equivalent” in the context of anIL3Rα antibody refers to an antibody that exhibits IL3Rα bindingactivity comparable to a reference IL3Rα antibody, as measured in an invivo and/or in vitro assay. “Functionally equivalent” in the context ofa cytotoxin refers to a compound that has cytotoxic activity comparableto that of a reference compound, as measured in an in vivo and/or invitro assay. In a specific embodiment, two IL3Rα antibodies arefunctionally equivalent if they bind to an overlapping epitope on IL3Rα,as measured by a competition assay described herein.

As used herein, the term “heterologous” in the context of an entity(e.g., a fusion protein) refers to an element that is part of an entity(e.g., a fusion protein) that is composed of one or more other elements,wherein the elements are not normally found or associated together. Forexample, in the context of a fusion protein, two or more amino acidsequences not normally found or associated together in nature arejoined, (by, e.g., conjugation).

As used herein, the phrase “human adult” refers to a human 18 years ofage or older.

As used herein, the phrase “human child” refers to a human between 24months of age and 18 years of age.

As used herein, the phrase “human infant” refers to a human less than 24months of age, preferably less than 12 months of age, less than 6 monthsof age, less than 3 months of age, less than 2 months of age, or lessthan 1 month of age.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing under whichnucleotide sequences at least 30% (preferably, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 99.5%) identical toeach other typically remain hybridized to each other. Such stringentconditions are known to those skilled in the art and can be found inCurrent Protocols in Molecular Biology, 1989, John Wiley & Sons, NewYork, Chapters 6.3.1-6.3.6. In one, non-limiting example, stringenthybridization conditions are hybridization at 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.1×SSC, 0.2% SDS at about 68° C. In a specific, non-limiting example,stringent hybridization conditions are hybridization in 6×SSC at about45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.(i.e., one or more washes at 50° C., 55° C., 60° C., or 65° C.). It isunderstood that the nucleic acids of the invention do not includenucleic acid molecules that hybridize, under these conditions, solely toa nucleotide sequence consisting of only A or T nucleotides. In aspecific embodiment, high-stringency conditions comprise hybridizationin a buffer consisting of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA,0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 100 μg/ml denatured salmon spermDNA, for 48 hours at 65° C., washing in a buffer composed of 2×SSC,0.01% PVP, 0.01% Ficoll, and 0.01% BSA, for 45 minutes at 37° C., andwashing in a buffer composed of 0.1×SSC, for 45 minutes at 50° C.

As used herein, the term “in combination” in the context of theadministration of a therapy to a subject refers to the use of more thanone therapy (e.g., prophylactic and/or therapeutic). The use of the term“in combination” does not restrict the order in which the therapies(e.g., a first and second therapy) are administered to a subject. Atherapy can be administered prior to (e.g., 1 minute, 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantlywith, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapy to a subject which had, has, or is susceptible to cancer.The therapies are administered to a subject in a sequence and within atime interval such that the therapies can act together. In a particularembodiment, the therapies are administered to a subject in a sequenceand within a time interval such that they provide an increased benefitthan if they were administered otherwise. Any additional therapy can beadministered in any order with the other additional therapy.

An “isolated” nucleic acid sequence or nucleotide sequence is one thatis separated from other nucleic acid molecules that are present in anatural source of the nucleic acid sequence or nucleotide sequence.Moreover, an “isolated” nucleic acid sequence or nucleotide sequence,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors when chemically synthesized.In certain embodiments, an “isolated” nucleic acid sequence ornucleotide sequence is a nucleic acid sequence or nucleotide sequencethat is recombinantly expressed in a heterologous cell.

As used herein, the terms “manage,” “managing,” and “management” in thecontext of the administration of a therapy to a subject refer to thebeneficial effects that a subject derives from a therapy (e.g., aprophylactic or therapeutic agent) or a combination of therapies, whilenot resulting in a cure of cancer. In certain embodiments, a subject isadministered one or more therapies (e.g., one or more prophylactic ortherapeutic agents) to “manage” cancer so as to prevent the progressionor worsening of the condition.

As used herein, the term “marker” in the context of a cell or tissue(e.g. a normal or cancer cell or tumor) means any antigen, molecule orother chemical or biological entity that is specifically found in or ona tissue that it is desired to identified or identified in or on aparticular tissue affected by a disease or disorder. In specificembodiments, the marker is a cell surface antigen that is differentiallyor preferentially expressed by specific cell types. For example, aleukemia cancer stem cell differentially expresses CD123 relative to anormal hematopoietic stem cell.

As used herein, the term “marker phenotype” in the context of a tissue(e.g., a normal or cancer cell or a tumor cell) means any combination ofantigens (e.g., receptors, ligands, and other cell surface markers),molecules, or other chemical or biological entities that arespecifically found in or on a tissue that it is desired to identify aparticular tissue affected by a disease or disorder. In specificembodiments, the marker phenotype is a cell surface phenotype. Inaccordance with this embodiment, the cell surface phenotype may bedetermined by detecting the expression of a combination of cell surfaceantigens. Non-limiting examples of cell surface phenotypes of cancerstem cells of certain tumor types include CD34⁺/CD38⁻, CD123⁺,CD44⁺/CD24⁻, CD133⁺, CD34⁺/CD10⁻/CD19⁻, CD138⁻/CD34⁻/CD19⁺, CD133⁺/RC2⁺,CD44⁺/α₂β₁ ^(hi)/CD133⁺, CLL-1, SLAMS, and other cancer stem cellsurface phenotypes mentioned herein, as well as those that are known inthe art.

“Percent Identity”: To determine the percent identity of amino acidsequences or their encoding nucleic acid sequences, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inthe sequence of a first amino acid or nucleic acid sequence for optimalalignment with a second amino acid or nucleic acid sequence). The aminoacid residues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions×100%). In one embodiment, the twosequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, “Methods for assessing the statistical significance ofmolecular sequence features by using general scoring schemes,” Proc.Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin andAltschul, “Applications and statistics for multiple high-scoringsegments in molecular sequences” Proc. Natl. Acad. Sci. USA 90:5873-5877(1993). Such an algorithm is incorporated into the BLASTN and BLASTXprograms of Altschul et al., “Basic local alignment search tool,” J.Mol. Biol. 215:403-410 (1990). BLAST nucleotide searches can beperformed with the BLASTN nucleotide program, with parameters set, e.g.,for score-100, word-size=12 to obtain nucleotide sequences homologous tonucleic acid molecules of the present invention. BLAST protein searchescan be performed with the BLASTX program, with parameters set, e.g., toscore-50, word-size=3 to obtain amino acid sequences homologous to aprotein molecule of the present invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al., “Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs,” Nucleic Acids Res. 25:3389-3402(1997). Alternatively, PSI-BLAST can be used to perform an iteratedsearch, which detects distant relationships between molecules (Id.).When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the defaultparameters of the respective programs (e.g., of BLASTX and BLASTN) canbe used (see, e.g., the National Center for Biotechnology Informationwebsite, http://www.ncbi.nlm.nih.gov/BLAST/). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, “Optimalalignments in linear space,” Comput. Appl. Biosci. 4:11-17 (1988). Suchan algorithm is incorporated in the ALIGN program (version 2.0), whichis part of the GCG® bioinformatics software package (Accelrys). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, gap length penalty of 12, and gap penalty of 4 canbe used.

The “percent identity” between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

As used herein, the phrase “pharmaceutically acceptable” means approvedby a regulatory agency of the federal or a state government, or listedin the United States Pharmacopeia, European Pharmacopeia, or othergenerally recognized pharmacopeia for use in animals, and moreparticularly, in humans.

As used herein, the terms “prevent,” “preventing” and “prevention” inthe context of the administration of a therapy to a subject refer to theprevention or inhibition of the recurrence, onset, and/or development ofa cancer or a symptom thereof in a subject resulting from theadministration of a therapy (e.g., a prophylactic or therapeutic agent),or a combination of therapies (e.g., a combination of prophylactic ortherapeutic agents). In some embodiments, such terms refer to one, two,three, or more results following the administration of one or moretherapies: (1) a stabilization, reduction or elimination of the cancerstem cell population, (2) a stabilization, reduction or elimination inthe cancer cell population, (3) an increase in response rate, (4) anincrease in the length or duration of remission, (5) a decrease in therecurrence rate of cancer, (6) an increase in the time to recurrence ofcancer, (7) an increase in the disease-free, relapse-free,progression-free, and/or overall survival of the patient, and (8) anamelioration of cancer-related symptoms and/or quality of life. Inspecific embodiments, such terms refer to a stabilization, reduction orelimination of the cancer stem cell population.

As used herein, the term “predetermined reference range” refers to areference range for the particular biological entity, e.g., cancer stemcell, for a subject or a population of subjects. Each laboratory mayestablish its own reference range for each particular assay, or astandard reference range for each assay may be made available and usedlocally, regionally, nationally, or worldwide or may bepatient-specific. In one specific embodiment, the term refers to areference range for the amount of cancer stem cells in a patient (e.g.,as determined by in vivo imaging) or a specimen from a patient. Inanother specific embodiment, the term refers to a reference range forthe amount of cancer cells in a patient (e.g. as described by in vivoimaging) or a specimen from a patient.

As used herein, the phrase “prophylactic agent” refers to any molecule,compound, and/or substance that is used for the purpose of preventingdisease, including but not limited to cancer, autoimmune disease, orallergic disease. Examples of prophylactic agents include, but are notlimited to, proteinaceous (such as immunoglobulins (e.g., multi-specificIgs, single chain Igs, Ig fragments, polyclonal antibodies and theirfragments, monoclonal antibodies and their fragments and bindingproteins), immunotoxins, chemospecific agents, chemotoxic agents (e.g.,anti-cancer agents), and small molecule drugs.

As used herein, the term “prophylactically effective regimen” refers toan effective regimen for dosing, timing, frequency and duration of theadministration of one or more therapies for the prevention of cancer ora symptom thereof. In a specific embodiment, the regimen achieves one,two, or three or more of the following results: (1) a stabilization,reduction or elimination of the cancer stem cell population; (2) astabilization, reduction or elimination in the cancer cell population;(3) a stabilization or reduction in the growth of a tumor or neoplasm;(4) an impairment in the formation of a tumor; (5) eradication, removal,or control of primary, regional and/or metastatic cancer; (6) areduction in mortality; (7) an increase in disease-free, relapse-free,progression-free, and/or overall survival, duration, or rate; (8) anincrease in the response rate, the durability of response, or number ofpatients who respond or are in remission; (9) a decrease inhospitalization rate; (10) a decrease in hospitalization lengths; (11)the size of the tumor is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%; (12) an increase in the number of patients inremission; (13) an increase in the length or duration of remission; (14)a decrease in the recurrence rate of cancer; (15) an increase in thetime to recurrence of cancer; and (16) an amelioration of cancer-relatedsymptoms and/or quality of life.

As used herein, the term “protocol” refers to a regimen for dosing andtiming of the administration of one or more agents and/or compositionsfor the prevention, treatment, and/or management of a disease or asymptom thereof. In certain embodiments, the term “protocol” refers tomethods of patient care that are associated with the administration ofan agent.

As used herein, the terms “purified” and “isolated” in the context of acompound or agent (including, e.g., proteinaceous agents such asantibodies) that is chemically synthesized refers to a compound or agentthat is substantially free of chemical precursors or other chemicalswhen chemically synthesized. In a specific embodiment, the compound oragent is 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 99% free (by dry weight)of other, different compounds or agents.

As used herein, the terms “purified” and “isolated” when used in thecontext of a compound or agent (including proteinaceous agents such asantibodies) that can be obtained from a natural source, e.g., cells,refers to a compound or agent that is substantially free ofcontaminating materials from the natural source, e.g., soil particles,minerals, chemicals from the environment, and/or cellular materials fromthe natural source, such as but not limited to cell debris, cell wallmaterials, membranes, organelles, the bulk of the nucleic acids,carbohydrates, proteins, and/or lipids present in cells. The phrase“substantially free of natural source materials” refers to preparationsof a compound or agent that has been separated from the material (e.g.,cellular components of the cells) from which it is isolated. Thus, acompound or agent that is isolated includes preparations of a compoundor agent having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dryweight) of cellular materials and/or contaminating materials.

As used herein, the phrase “small molecule(s)” and analogous termsinclude, but are not limited to, peptides, peptidomimetics, amino acids,amino acid analogs, polynucleotides, polynucleotide analogs,nucleotides, nucleotide analogs, and other organic and inorganiccompounds (i.e., including hetero-organic and organometallic compounds)having a molecular weight less than about 10,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 5,000grams per mole, organic or inorganic compounds having a molecular weightless than about 1,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 500 grams per mole, organic orinorganic compounds having a molecular weight less than about 100 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

As used herein, the term “stabilizing” and analogous terms, when used inthe context of a cancer stem cell population or cancer cell population,refer to the prevention of an increase in the cancer stem cellpopulation or cancer cell population, respectively. In other words, theamount of cancer stem cells or the amount of cancer cells that a canceris composed of is maintained, and does not increase, or increases byless than 10%, preferably less than 5%.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the term “subject” refers to an animal,preferably a mammal such as a non-primate (e.g., cows, pigs, horses,cats, dogs, rats etc.) and a primate (e.g., monkey and human), and mostpreferably a human. In some embodiments, the subject is a non-humananimal such as a farm animal (e.g., a horse, pig, or cow) or a pet(e.g., a dog or cat). In a specific embodiment, the subject is anelderly human. In another embodiment, the subject is a human adult. Inanother embodiment, the subject is a human child. In yet anotherembodiment, the subject is a human infant.

As used herein, the term “therapeutic agent” refers to any molecule,compound, and/or substance that is used for the purpose of treatingand/or managing cancer. Examples of therapeutic agents include, but arenot limited to, proteins, immunoglobulins (e.g., multi-specific Igs,single chain Igs, Ig fragments, polyclonal antibodies and theirfragments, monoclonal antibodies and their fragments), antibodyconjugates or antibody fragment conjugates, peptides (e.g., peptidereceptors, selectins), binding proteins, chemospecific agents,chemotoxic agents (e.g., anti-cancer agents), radiation, chemotherapy,anti-angiogenic agents, and small molecule drugs. Therapeutic agents maybe a(n) anti-angiogenesis therapy, targeted therapy, radioimmunotherapy,small molecule therapy, biologic therapy, epigenetic therapy, toxintherapy, differentiation therapy, pro-drug activating enzyme therapy,antibody therapy, chemotherapy, radiation therapy, hormonal therapy,immunotherapy, or protein therapy.

As used herein, the term “therapeutically effective regimen” refers to aregimen for dosing, timing, frequency, and duration of theadministration of one or more therapies for the treatment and/ormanagement of cancer or a symptom thereof. In a specific embodiment, theregimen achieves one, two, three, or more of the following results: (1)a stabilization, reduction or elimination of the cancer stem cellpopulation; (2) a stabilization, reduction or elimination in the cancercell population; (3) a stabilization or reduction in the growth of atumor or neoplasm; (4) an impairment in the formation of a tumor; (5)eradication, removal, or control of primary, regional and/or metastaticcancer; (6) a reduction in mortality; (7) an increase in disease-free,relapse-free, progression-free, and/or overall survival, duration, orrate; (8) an increase in the response rate, the durability of response,or number of patients who respond or are in remission; (9) a decrease inhospitalization rate; (10) a decrease in hospitalization lengths; (11)the size of the tumor is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%; (12) an increase in the number of patients inremission; (13) an increase in the length or duration of remission; (14)a decrease in the recurrence rate of cancer; (15) an increase in thetime to recurrence of cancer; and (16) an amelioration of cancer-relatedsymptoms and/or quality of life.

As used herein, the terms “therapies” and “therapy” can refer to anymethod(s), composition(s), and/or agent(s) that can be used in thetreatment of a cancer or one or more symptoms thereof. In certainembodiments, the terms “therapy” and “therapies” refer to chemotherapy,radiation therapy, radioimmunotherapy, hormonal therapy, targetedtherapy, toxin therapy, pro-drug activating enzyme therapy, proteintherapy, antibody therapy, small molecule therapy, epigenetic therapy,demethylation therapy, histone deacetylase inhibitor therapy,differentiation therapy, antiangiogenic therapy, biological therapyincluding immunotherapy and/or other therapies useful in the treatmentof a cancer or one or more symptoms thereof.

As used herein, the terms “treat”, “treatment”, and “treating” in thecontext of the administration of a therapy to a subject refer to thereduction or inhibition of the progression and/or duration of cancer,the reduction or amelioration of the severity of cancer, and/or theamelioration of one or more symptoms thereof resulting from theadministration of one or more therapies. In a specific embodiment, apatient that is at a high risk for developing cancer is treated. Inspecific embodiments, such terms refer to one, two, or three or moreresults following the administration of one, two, three or moretherapies: (1) a stabilization, reduction or elimination of the cancerstem cell population; (2) a stabilization, reduction or elimination inthe cancer cell population; (3) a stabilization or reduction in thegrowth of a tumor or neoplasm; (4) an impairment in the formation of atumor; (5) eradication, removal, or control of primary, regional and/ormetastatic cancer; (6) a reduction in mortality; (7) an increase indisease-free, relapse-free, progression-free, and/or overall survival,duration, or rate; (8) an increase in the response rate, the durabilityof response, or number of patients who respond or are in remission; (9)a decrease in hospitalization rate; (10) a decrease in hospitalizationlengths; (11) the size of the tumor is maintained and does not increaseor increases by less than 10%, preferably less than 5%, preferably lessthan 4%, preferably less than 2%; (12) an increase in the number ofpatients in remission; (13) an increase in the length or duration ofremission; (14) a decrease in the recurrence rate of cancer; (15) anincrease in the time to recurrence of cancer; and (16) an ameliorationof cancer-related symptoms and/or quality of life. In certainembodiments, such terms refer to a stabilization or reduction in thecancer stem cell population. In some embodiments, such terms refer to astabilization or reduction in the growth of cancer cells. In someembodiments, such terms refer to a stabilization or reduction in thecancer stem cell population and a reduction in the cancer cellpopulation. In some embodiments, such terms refer to a stabilization orreduction in the growth and/or formation of a tumor. In someembodiments, such terms refer to the eradication, removal, or control ofprimary, regional, or metastatic cancer (e.g., the minimization or delayof the spread of cancer). In some embodiments, such terms refer to areduction in mortality and/or an increase in survival rate of a patientpopulation. In further embodiments, such terms refer to an increase inthe response rate, the durability of response, or number of patients whorespond or are in remission. In some embodiments, such terms refer to adecrease in hospitalization rate of a patient population and/or adecrease in hospitalization length for a patient population.

As used herein the phrases “VH domain,” “VH domain,” “VH region” referto the variable region of the heavy chain of an antibody which iscomposed of three CDRs and the framework regions. As used herein, thephrases “VL domain,” “VL domain” and “VL region” refer to the variableregion of the light chain of an antibody which is composed of three CDRsand the framework regions.

Concentrations, amounts, cell counts, percentages, and other numericalvalues may be presented herein in a range format. It is to be understoodthat such range format is used merely for convenience and brevity andshould be interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D. FIG. 1A shows the DNA nucleotide sequence of the V_(H)domain (SEQ ID NO: 1) of the 26292 monoclonal or scFv antibody. FIG. 2Bshows the amino acid sequence of the V_(H) domain (SEQ ID NO: 2) of the26292 monoclonal or scFv antibody with the V_(H) CDR1 (SEQ ID NO: 3),V_(H) CDR2 (SEQ ID NO: 4), and v CDR3 (SEQ ID NO: 5) underlined,starting in order from V_(H) CDR1 at the far left. The framework regionsare the regions not underlined. FIG. 2C shows the nucleotide sequence ofthe V_(L) domain (SEQ ID NO: 6) of the 26292 monoclonal scFv antibody.FIG. 2D shows the amino acid sequence of the V_(L) domain (SEQ ID NO: 7)of the 26292 monoclonal or scFv antibody with the V_(L) CDR1 (SEQ ID NO:8), V_(L) CDR2 (SEQ ID NO: 9), and VL CDR3 (SEQ ID NO: 10) underlined,starting in order from V_(L) CDR1 at the far left. The framework regionsare the regions not underlined.

FIGS. 2A-2D. FIG. 2A shows the DNA nucleotide sequence of the V_(H)domain (SEQ ID NO: 11) of the 32703 monoclonal or scFv antibody. FIG. 2Bshows the amino acid sequence of the V_(H) domain (SEQ ID NO: 12) of the32703 monoclonal or scFv antibody with the V_(H) CDR1 (SEQ ID NO:13),V_(H)CDR2 (SEQ ID NO:14), and V_(H) CDR3 (SEQ ID NO: 15) underlined,starting in order from V_(H) CDR1 at the far left. The framework regionsare the regions not underlined. FIG. 2C shows the DNA nucleotidesequence of the V_(L) domain (SEQ ID NO:16) of the 32703 monoclonal orscFv antibody. FIG. 2D shows the amino acid sequence of the V_(L) domain(SEQ ID NO: 17) of the 32703 monoclonal or scFv antibody with the V_(L)CDR1 (SEQ ID NO: 18), V_(L) CDR2 (SEQ ID NO: 19), and V_(L) CDR3 (SEQ IDNO: 20) underlined, starting in order from VL CDR1 at the far left. Theframework regions are the regions not underlined.

FIGS. 3A-3D. FIG. 3A shows the DNA nucleotide sequence of the V_(H)domain (SEQ ID NO: 21) of the 32701 monoclonal or scFv antibody. FIG. 3Bshows the amino acid sequence of the V_(H) domain (SEQ ID NO: 22) of the32701 monoclonal or scFv antibody with the V_(H) CDR1 (SEQ ID NO: 23),V_(H) CDR2 (SEQ ID NO: 24), and V_(H) CDR3 (SEQ ID NO: 25) underlined,starting in order from V_(H) CDR1 at the far left. The framework regionsare the regions not underlined. FIG. 3C shows the DNA nucleotidesequence of the V_(L) domain (SEQ ID NO: 26) of the 32701 monoclonal orscFv antibody. FIG. 3D shows the amino acid sequence of the V_(L) domain(SEQ ID NO: 27) of the 32701 monoclonal or scFv antibody with the V_(L)CDR1 (SEQ ID NO: 28), V_(L) CDR2 (SEQ ID NO: 29), and V_(L) CDR3 (SEQ IDNO: 30) underlined, starting in order from V_(L) CDR1 at the far left.The framework regions are the regions not underlined.

FIGS. 4A-4D. FIG. 4A shows the DNA nucleotide sequence of the VH domain(SEQ ID NO: 31) of the 32716 monoclonal or scFv antibody. FIG. 4B showsthe amino acid sequence of the V_(H) domain (SEQ ID NO: 32) of the 32716monoclonal or scFv antibody with the V_(H) CDR1 (SEQ ID NO: 33), V_(H)CDR2 (SEQ ID NO: 34), and VH CDR3 (SEQ ID NO: 35) underlined, startingin order from V_(H) CDR1 at the far left. The framework regions are theregions not underlined. FIG. 4C shows the DNA nucleotide sequence of theshows the V_(L) domain (SEQ ID NO: 36) of the 32716 monoclonal or scFvantibody. FIG. 4D shows the amino acid sequence of the V_(L) domain (SEQID NO: 37) of the 32716 monoclonal or scFv antibody with the V_(L) CDR1(SEQ ID NO: 38), V_(L) CDR2 (SEQ ID NO: 39), and V_(L) CDR3 (SEQ ID NO:40) underlined, starting in order from V_(L) CDR1 at the far left. Theframework regions are the regions not underlined.

FIG. 5A-5B. FIG. 5A shows an alignment of the V_(H) amino acid sequencesof the 32703 monoclonal or scFv antibody, 26292 monoclonal or scFvantibody, the 32716 monoclonal or scFv antibody, the 32701 monoclonal orscFv antibody, and germline sequences. FIG. 5B shows an alignment of theV_(L) amino acid sequences of the 32703 monoclonal or scFv antibody,26292 monoclonal or scFv antibody, the 32716 monoclonal or scFvantibody, the 32701 monoclonal or scFv antibody, and germline sequences.

FIGS. 6A-6B. FACS analysis of binding of anti-CD123 antibodies to TF-1and SDS-PAGE analysis of purified immunotoxins. FIG. 6A: TF-1 werestained with an isotype control MOPC-21 or anti-CD 123 antibodies at 0.5μg/ml. The fluorescence units after subtraction of the isotype controlare shown. FIG. 6B: 4 μg of immunotoxin per lane. Lane “M”, PrecisionPlus protein marker (Bio-Rad, kDa); lane “92”, 26292(Fv)-PE38; lane“01”, 32701(Fv)-PE38; and lane “16”, 32716(Fv)-PE38.

FIGS. 7A-7D. Binding ability, cytotoxic activity and stability ofanti-CD123 immunotoxins. FIG. 7A: FACS analysis of binding of26292(Fv)-PE38, 32701(Fv)-PE38 and 32716(Fv)-PE38 to TF-1. Allimmunotoxins used were at 60 nM. FIG. 7B: Apparent affinity of26292(Fv)-PE38 on TF-1. Binding saturation curve and nonlinearregression analysis were produced with Graph Pad Prism (Graph PadSoftware, Inc). FIG. 7C: Cytotoxicity of 26292(Fv)-PE38, 32701(Fv)-PE38and 32716(Fv)-PE38 on TF-1. FIG. 7D: Stability of immunotoxin26292(Fv)-PE38. 200 ng/ml of immunotoxin was incubated at 4° C. (•) or37° C. (▪) for 1, 3 and 6 hours. The activity at time zero is set as100%.

FIG. 8. The expression of CD123 on leukemia cell lines. QuantiBRITE PEBeads were used as the PE fluorescence standard. Cell lines were stainedwith isotype control-PE (open region) or 9F5-PE (filled region) at 10μg/ml.

FIG. 9. The cytotoxicity of 26292(Fv)-PE38 (▴), 26292(Fv)-PE38-KDEL (▪),and HB21(Fv)-PE40 (•) on leukemia cell lines.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical compositions comprising anantibody that binds to the IL3Rα chain (preferably, the human IL3Rαchain), and a pharmaceutically acceptable carrier or excipient. In oneaspect, the invention provides pharmaceutical compositions comprising anantibody that binds to the IL3Rα chain in an amount effective to reducethe quantity, number, amount or percentage of cancer stem cells and/orcancer cells in an animal with or animal model for myeloid leukemia oranother cancer associated with IL3Rα-expressing cells by about 25%,about 30%, 35%, about 40%, about 45%, about 50%, about 65%, about 75% ormore relative to a negative control. In a specific embodiment, theantibody incorporated into a pharmaceutical composition has adissociation constant (Kd) of less than 4 nM on cells expressing theIL3Rα chain (e.g., erytholeukemic cells, myeloid cells or myeloblasticcells). In another embodiment, the antibody incorporated into apharmaceutical composition comprises a variable heavy (VH) domain havingthe amino acid sequence of SEQ ID NO:2, 12, 22, or 32, and/or a variablelight (VL) domain having the amino acid sequence of SEQ ID NO: 7, 17, 27or 37. In another embodiment, the antibody incorporated into apharmaceutical composition comprises one or more of the VHcomplementarity determining regions (CDRs) shown in FIGS. 1B, 2B, 3Band/or 4B and/or a VL domain comprising one or more of the VL CDRs shownin FIGS. 1D, 2D, 3D and/or 4D. In another embodiment, the antibodyincorporated into a pharmaceutical composition competes with an antibodycomprising a VH domain having the amino acid sequence of SEQ ID NO:2,12, 22, or 32 and a VL domain having the amino acid sequence of SEQ IDNO: 7, 17, 27 or 37 for binding to IL3Rα chain. In some embodiments, apharmaceutical composition of the invention comprises an agent inaddition to an antibody of the invention.

The present invention also provides methods for inhibiting theproliferation or reducing an IL3Rα-expressing cell population, themethods comprising contacting a population of cells comprisingIL3Rα-expressing cells with an antibody that binds to the IL3Rα chain.In a specific embodiment, the present invention provides methods forinhibiting the proliferation or reducing the population of cancer stemcells expressing IL3Rα, the methods comprising contacting theIL3Rα-expressing cancer stem cell population with an antibody that bindsto the IL3Rα chain. In another embodiment, the present inventionprovides methods for inhibiting the proliferation or reducing thepopulation of cancer cells expressing IL3Rα, the methods comprisingcontacting the IL3Rα-expressing cancer cell population with an antibodythat binds to the IL3Rα chain. In yet another embodiment, the presentinvention provides methods for inhibiting the proliferation or reducingthe population cancer cells and cancer stem cells expressing IL3Rα, themethods comprising contacting a population of cells comprisingIL3Rα-expressing cancer cells and/or IL3Rα-expressing cancer stem cellswith an antibody that binds to the IL3Rα chain. In certain embodiments,the antibody reduces the quantity, number, amount or percentage ofcancer stem cells and/or cancer cells by at least 25%, at least 30%, atleast 40%, at least 50%, at least 65%, at least 75%, or at least 85% inan animal with or animal model for myeloid leukemia or another cancerassociated with IL3Rα-expressing cells relative to a negative control.

The present invention also provides methods for preventing, treatingand/or managing a disorder associated with IL3Rα-expressing cells (e.g.,a hematologic cancer), the methods comprising administering to a subjectin need thereof an antibody that binds to the IL3Rα chain. In a specificembodiment, the present invention provides a method for preventing,treating and/or managing a disorder associated with IL3Rα-expressingcells (e.g., a hematologic cancer), the method comprising administeringto a human subject in need thereof an effective amount of an antibodythat binds to the IL3Rα chain, and in some embodiments, an effectiveamount of another therapy. Non-limiting examples of disorders associatedwith IL3Rα-expressing cells include autoimmune disorders (such aslupus), inflammatory disorders (such as allergies and asthma) andcancers (such as hematological cancers). In some embodiments, thedisorder associated with IL3Rα-expressing cells is not myeloid leukemia.

The present invention provides methods for preventing relapse of cancerassociated with IL3Rα-expressing cells, the methods comprisingadministering to a subject in need thereof an antibody that binds to theIL3Rα chain. In a specific embodiment, the present invention providesmethods for preventing relapse of cancer associated withIL3Rα-expressing cells, the methods comprising administering to a humansubject in need thereof an effective amount of an antibody that binds tothe IL3Rα chain, and in some embodiments, an effective amount of anothertherapy. In some embodiments, the cancer associated withIL3Rα-expressing cells is not myeloid leukemia.

The present invention provides methods for preventing, treating and/ormanaging cancer associated with IL3Rα-expressing cells (e.g., ahematologic cancer), the method comprising administering to a humansubject in need thereof an antibody that binds to the IL3Rα chain in anamount effective to reduce the quantity, number, amount or percentage ofcancer stem cells and/or cancer cells by at least 25%, at least 30%, atleast 40%, at least 50%, at least 65%, at least 75%, or at least 85% inan animal with or animal model for myeloid leukemia or another cancerassociated with IL3Rα-expressing cells relative to a negative control.

The present invention provides antibody conjugates comprising anantibody that binds to the alpha chain of the IL-3 receptor alphasubunit (IL3Rα, CD123, preferably, the human IL3Rα) linked to acytotoxic agent or other moiety, and compositions comprising suchconjugates and uses of such conjugates. In one aspect, the presentinvention provides an antibody conjugate comprising an antibody thatbinds to the IL3Rα chain linked to a cytotoxic agent, wherein theimmunotoxin has an IC50 of less than 40 ng/ml on cells expressing theIL3Rα chain. In another aspect, the present invention provides anantibody conjugate comprising an antibody that binds to the IL3Rα chainlinked to a cytotoxic agent, wherein the antibody comprises a VH domainhaving the amino acid sequence shown in FIG. 1B, 2B, 3B or 4B and/or aVL domain having the amino acid sequence shown in FIG. 1D, 2D, 3D or 4D.In another aspect, the present invention provides an antibody conjugatecomprising an antibody that binds to the IL3Rα chain linked to acytotoxic agent, wherein the antibody comprises one or more of the VHCDRs shown in FIGS. 1B, 2B, 3B and/or 4B and/or one or more of the VLCDRs shown in FIGS. 1D, 2D, 3D and/or 4D. In yet another aspect, thepresent invention provides an antibody conjugate comprising an antibodythat binds to the IL3Rα chain linked to a cytotoxic agent, wherein theantibody competes with an antibody comprising a variable heavy (VH)domain having the amino acid sequence of SEQ ID NO:2, 12, 22, or 32,and/or a variable light (VL) domain having the amino acid sequence ofSEQ ID NO: 7, 17, 27 or 37 for binding to IL3Rα chain.

The present invention provides compositions comprising an antibodyconjugate of the invention, and a carrier or excipient. In a specificembodiment, the present invention provides pharmaceutical compositionscomprising an antibody conjugate of the invention, and apharmaceutically acceptable carrier or excipient. In a specificembodiment, the invention provides pharmaceutical compositionscomprising an antibody conjugate of the invention in an amount effectiveto reduce the quantity, number, amount or percentage of cancer stemcells and/or cancer cells in an animal with or animal model for myeloidleukemia or another cancer by about 25%, about 30%, 35%, about 40%,about 45%, about 50%, about 65%, about 75% or more relative to anegative control. In some embodiments, the compositions of the inventioncomprise an agent in addition to the antibody conjugate.

The present invention provides methods for inhibiting the proliferation,or reducing or eradicating the IL3Rα-expressing cell population, themethods comprising contacting a population of cells comprisingIL3Rα-expressing cells with an antibody conjugate of the invention. Thepresent invention also provides methods for preventing, treating and/ormanaging a disorder associated with IL3Rα-expressing cells (e.g., ahematologic cancer), the methods comprising administering to a subjectin need thereof an antibody conjugate of the invention. The presentinvention further provides methods for ex vivo purging of bone marrow orperipheral blood to remove cells that express IL3Rα such that the purgedbone marrow or peripheral blood is suitable, e.g., for autologous stemcell transplantation to restore hematopoietic function.

5.1 Anti-IL3Rα Antibody Conjugates

The present invention provides antibody conjugates that bind to theIL3Rα chain (preferably, the human IL3Rα chain). In some embodiments,the antibody conjugates of the present invention comprise an antibodythat binds to the IL3Rα chain expressed on the surface of cellsconjugated to a cytotoxic agent or other moiety (e.g., an anticellularmoiety). In some embodiments, the antibody conjugates of the presentinvention comprise an antibody that binds to the extracellular domain ofthe IL3Rα chain conjugated to a cytotoxic agent or other moiety (e.g.,an anticellular moiety). In a specific embodiment, the antibodyconjugate of the invention is an immunotoxin. In one embodiment, theantibody is conjugated to a cytotoxic agent or otherwise anticellularagent, either directly or through a chemical linker. In anotherembodiment, the antibody is linked to the cytotoxic agent or otherwiseanticellular moiety through a chemical (covalent) bond, such as apeptide bond (with or without a peptide linker), disulfide bond, orsterically hindered disulfide bond. The antibody can be linked at itsamino terminus or its carboxyl terminus to the cytotoxic agent orotherwise anticellular moiety. Alternatively, the antibody can replace adomain of the cytotoxic agent or otherwise anticellular moiety that isnot required for cytotoxicity so long as antibody retains itsspecificity for the IL3Rα chain.

In certain embodiments, an antibody conjugate comprises an IL3Rαantibody linked via a peptide linker (also referred to as a spacer) to acytotoxic agent or an otherwise anticellular moiety. The linker for theconjugate may be 5, 8, 10, 12, 15, 25, 50, or 75 amino acids in length,but the length may otherwise vary to provide optimal binding of theconjugate to IL3Rα. In a specific embodiment, the peptide linker is 6 or7 amino acids long. In another embodiment, the amino terminus of anIL3Rα antibody is attached to a cytotoxic agent or an otherwiseanticellular moiety through the peptide linker Ser-(Gly)4-Ser. Inanother embodiment, the carboxyl terminus of an IL3Rα antibody is linkedto a cytotoxic agent or an otherwise anticellular moiety through aLys-Ala-Ser-Gly-Gly-Pro-Glu linker. The constituent amino acids of aspacer may be selected to influence some property of the conjugate, suchas its folding, net charge, or hydrophobicity. Linker molecules arecommonly known in the art and described in Denardo et al., 1998, ClinCancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug. Chem. 10:553;and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50 each incorporatedby reference in their entireties.

An antibody conjugate of the invention may be composed of one or twopolypeptide chains, together comprising the antibody moiety and thecytotoxic agent or anticellular moiety. In one embodiment, the cytotoxicagent is attached to the variable heavy (V_(H)) region of an Fv antibodyfragment against IL3Rα, where the V_(H) region is bound by an amino acidlinker to the variable light (V_(L)) chain region. In anotherembodiment, the cytotoxic agent is attached to the V_(H) region of an Fvantibody against IL3Rα, where the V_(H) region is bound to a V_(L) chainregion through at least one disulfide linkage (e.g., formed betweenrespective cysteines in each chain). Disulfide linked Fv chains may havea reduced tendency to aggregate, show a generally longer serumhalf-life, and are said to be “stabilized.” Thus, a disulfide-stabilizedantibody-cytotoxin conjugate comprises at least two polypeptides linkedby at least one disulfide linkage. The two polypeptides can be separatedby a termination codon and downstream initiation codon and ribosomebinding site, so that the chains are encoded as separate open readingframes, or they can be joined by a peptide linker. In anotherembodiment, the cytotoxic agent is attached to the V_(H) region of an Fvantibody against IL3Rα, where the V_(H) region is bound through apeptide linker and at least one disulfide bond to a V_(L) chain region.In a specific embodiment, the cytotoxic agent is attached to V_(L)region of an Fv antibody against IL3Rα, where the V_(L) region is boundby a linker to the V_(H) chain region. In another embodiment, thecytotoxic agent is attached to the V_(L) region of an Fv antibodyagainst IL3Rα, where the V_(L) region is bound through at least onedisulfide bond to a V_(H) chain region. In another embodiment, thecytotoxic agent is attached to the V_(L) region of an Fv antibodyagainst IL3Rα, where the V_(L) region is bound through a peptide linkerand at least one disulfide bond to a V_(H) chain region. In yet anotherembodiment of the invention, the V_(L) and V_(H) sequences will befollowed respectively by part or all of the light and heavy chainconstant regions, e.g., the whole kappa light chain constant region andthe C_(H1) domain of the heavy chain constant region, with or withoutthe heavy chain hinge domain. Thus, the antibody segments and genesencoding the cytotoxic agent may occur in any order on a single plasmid,or may be expressed separately from separate plasmids. For example, inanother embodiment of the invention, the V_(L) gene and any light chainconstant region will be on one plasmid, while the V_(H) gene, any heavychain constant region, and the gene for a proteinaceous cytotoxin willbe on a second plasmid. In either case, the V_(L) and/or V_(H) genes maybe preceded by a signal sequence that directs the secretion of therecombinant fusion protein from the cell. See, e.g., U.S. Pat. Nos.6,147,203, 6,074,644 and 6,051,405 which are referenced herein in theirentirety.

In another embodiment, the polypeptide chains expressed by the plasmidmay be sequestered in inclusion bodies that are retained within thecell. The polypeptides may be expressed in a variety of expressionsystems that are routinely available to one skilled in the art,including bacterial expression systems such as E. coli and yeastexpression systems, such as Pichia. See, e.g., Vrieto et al., 2004,Protein Expression and Purification 33: 1123-133, which is incorporatedherein by reference in its entirety, for methods of expressingpolypeptides sequestered in inclusion bodies.

In another embodiment, this invention provides for single chain antibodyconjugates, in which the antibody comprises the V_(L) or V_(H) regionsalone, rather than as components of Fv fragments. The amino terminus orcarboxyl terminus of the variable chain is then conjugated to a selectedcytotoxic agent, such conjugation may be through a peptide linker. See,e.g., U.S. Pat. No. 6,074,644, which is referenced herein in itsentirety.

Antibodies, or the encoded antibodies, cytotoxic agents, or antibodyconjugates may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. A polynucleotide encoding an IL3Rα antibody may berecombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more cytotoxin molecules.

The invention provides nucleic acids comprising nucleotide sequencesencoding an antibody conjugate of the invention. The invention alsoprovides nucleic acids comprising a nucleotide sequence that encode theantibody moiety and/or the proteinaceous cytotoxic agent of an antibodyconjugate of the invention. Thus, for example, in one embodiment, theinvention provides for a nucleic acid comprising a nucleotide sequenceencoding an antibody conjugate comprising a V_(H) region of an Fvfragment attached to a proteinaceous cytotoxic agent (e.g., Pseudomonasexotoxin). The nucleotides that encode the V_(H) region are linked tothe nucleotides that encode the V_(L) region through nucleotides thatencode a peptide linker. Alternatively, or in combination, the encodedV_(H) region contains cysteine residues that form disulfide linkageswith a V_(L) region of an Fv fragment. In another embodiment, theinvention provides for nucleic acids comprising nucleotide sequenceencoding an antibody conjugate of the invention in which the V_(L) chainis substituted for the V_(H) chain and vice versa. In anotherembodiment, the invention provides for nucleic acids comprising anucleotide sequence encoding an antibody conjugate, in which theantibody comprises the V_(L) or V_(H) region alone. See, e.g., U.S. Pat.No. 6,074,644, which is referenced herein in its entirety.

In certain embodiments, an antibody conjugate of the invention competeswith IL-3 for binding to the IL3Rα chain. In a specific embodiment, anantibody conjugate of the invention competes with IL-3 for binding toIL3Rα-expressing cells. In certain embodiments, an antibody conjugateprevents IL-3 receptor signal transduction.

In some embodiments, an antibody conjugate of the invention binds to anepitope on IL3Rα that results in selective and potent cell killing, asassessed by a cytotoxicity assay. In certain embodiments, binding of anantibody conjugate to its epitope on IL3Rα is followed byinternalization of the antibody conjugate-IL3Rα complex. In a particularembodiment, the cytotoxicity of a particular antibody-cytotoxinconjugate is measured by a cell viability assay, using cell lines thatexpress IL3Rα (e.g., TF-1, Molm-13, or Molm-14). In a particularembodiment, cell viability is tested using an assay in which cells areseeded into 96-well plates at a concentration of 2×10⁴ of cells/well.Serial dilutions of antibody conjugates in 0.2% human serum albumin(HSA) are added to the cells, resulting in final concentrations rangingfrom 0.1 to 1000 ng/ml in 150 pl. After incubation for 48 hours, 10 μlof WST-8 (Dojindo Molecular Technologies) is added to each well, and theincubation is carried out for 4 hours at 37° C. The absorbance of thesample at 450 nm is measured with a reference wavelength of 650 nm.Cytotoxicity is defined by IC₅₀, 50% inhibition of cell viability, whichis midway between the level of viability in the absence of antibodyconjugate and that in the presence of 10 μg/ml cycloheximide.

The present invention also provides for panels of antibody conjugatesthat bind to the IL3Rα chain. In specific embodiments, the inventionprovides for panels of antibody conjugates having different affinitiesfor the IL3Rα chain, different specificities for the IL3Rα chain, and/ordifferent dissociation rates. The invention provides panels of at least10, preferably at least 25, at least 50, at least 75, at least 100, atleast 125, at least 150, at least 175, at least 200, at least 250, atleast 300, at least 350, at least 400, at least 450, at least 500, atleast 550, at least 600, at least 650, at least 700, at least 750, atleast 800, at least 850, at least 900, at least 950, or at least 1000antibodies. Panels of antibody conjugates can be used, for example, in96 well plates for assays such as ELISAs and cytotoxicity assays.

5.2 Antibodies that Bind to IL3Rα

It should be recognized that antibodies that bind to IL3Rα antigens areknown in the art.

Set forth below is a more detailed description of the anti-IL3Rαantibodies encompassed within the various aspects of the invention. Suchantibodies can be conjugated to cytotoxic agents or other moieties(e.g., an anticellular moiety) and used as potent and specificimmunotoxins. Alternatively, the unconjugated antibodies (i.e., nakedantibodies) can be used as prophylactic and therapeutic agents.

The present invention provides methods for assessing specificity,affinity, and cytotoxicity of antibodies and antibody conjugates, suchthat the antibodies and antibody conjugates of the invention may beuseful to specifically target and impair cells expressing IL3Rα. Thepresent invention provides antibodies that may differentially orpreferentially bind to one or more epitopes on IL3Rα. The presentinvention provides methods for comparing various anti-IL3Rα antibodieswith respect to their differential epitope binding. The presentinvention provides methods for assessing the affinity of variousanti-IL3Rα antibodies for their specific IL3Rα antigen on cells.

Antibodies of the invention include, but are not limited to, monoclonalantibodies, monospecific antibodies, polyclonal antibodies,multispecific antibodies, diabolizes, triabodies, tetrabodies, humanantibodies, humanized antibodies, camelized antibodies, chimericantibodies, single chain antibodies, single domain antibodies, Fabfragments, F(ab′) fragments, F(ab′)2 fragments, Fv fragments (i.e., thesmallest functional module of an antibody), single chain Fvs (scFv),disulfide-stabilized Fvs (dsFv), Fd, V_(H), V_(L), V_(α), V_(β), andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), intrabodies, and epitope-bindingfragments of any of the above. In some embodiments, the antibodies aremonoclonal antibodies. In other embodiments, the antibodies are Fvfragments, including V_(H) and V_(L) regions.

In particular, antibodies of the present invention includeimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that binds to an IL3Rα antigen. The immunoglobulinmolecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD,IgA, and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂), orsubclass of immunoglobulin molecule. In a specific embodiment, anantibody of the invention is an IgG antibody. In another specificembodiment, an antibody of the invention is not an IgA antibody.

The antibodies of the invention may be from any animal origin includingbirds and mammals (e.g., human, mouse, donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken). Preferably, the antibodies of theinvention are human or humanized monoclonal antibodies. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from mice that express antibodies from humangenes.

The antibodies of the present invention may be monospecific, bispecific,trispecific, or of greater multispecificity. Multispecific antibodiesmay be specific for different epitopes of an IL3Rα polypeptide or may bespecific for both an IL3Rαpolypeptide as well as for a heterologousepitope, such as a heterologous polypeptide or solid support material,as long as such epitopes are not found in human tissues. See, e.g., PCTpublications WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793;Tutt, et al., J. Immunol. 147:60-69(1991); U.S. Pat. Nos. 4,474,893,4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al., J.Immunol. 148:1547-1553 (1992).

In one embodiment, antibodies of the invention bind to cells thatexpress IL3Rα chain (preferably, human IL3Rα chain). In a specificembodiment, antibodies of the invention bind to a fragment of the IL3Rαchain comprising 8 to 285, 8 to 275, 8 to 250, 8 to 200, 8 to 175, 8 to150, 8 to 100, 8 to 50, 8 to 25, 15 to 50, 15 to 75, 15 to 100 or 15 to150 amino acids of the extracellular domain of the mature IL3Rα chain(preferably, the mature human IL3Rα chain)s. In another embodiment, theantibodies of the invention bind to the extracellular domain of thehuman IL3Rα chain or a fragment thereof.

An antibody of the invention may be composed of one or two polypeptidechains. In one embodiment, an Fv antibody that binds to the IL3Rα chainis composed of a single polypeptide chain, where the V_(H) region isbound by an amino acid linker to the V_(L) chain region. In a preferredembodiment, such scFvs are stable at 37° C. for about 2 minutes, 5minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours,12 hours, 14 hours, 24 hours, 48 hours, 1 week, 2 weeks, 1 month, 3months, 6 months, 1 year or longer as assessed by a technique known toone of skill in the art or described herein. In another embodiment, theV_(H) region of an Fv antibody that binds to the IL3Rα chain is bound toa V_(L) chain region through at least one disulfide linkage (e.g.,formed between respective cysteines in each chain). In certainembodiments, the disulfide linked Fv chains have a reduced tendency toaggregate as measured by, e.g., HPLC and have a longer serum half-life.Thus, in a specific embodiment, a disulfide-stabilized Fv (dsFv)antibody comprises at least two polypeptides linked by at least onedisulfide linkage. The two polypeptides can be separated by atermination codon and downstream initiation codon and ribosome bindingsite, so that the chains are encoded as separate open reading frames, orthey can be additionally joined by a peptide linker. In order to providedisulfide covalent bonds between the V_(H) and V_(L) chains of dsFvfragments, cysteine residues are necessary. Cysteine residues can beintroduced in the proper position of each V_(H) and V_(L), determined byalignment to reference sequences, by standard molecular biologytechniques (e.g., site directed mutagenesis). See Pastan et al., U.S.Pat. No. 6,147,203, which is incorporate by reference herein in itsentirety, especially columns 5-7.

In another embodiment, the V_(L) and V_(H) sequences will be followedrespectively by part or all of the light and heavy chain constantregions, e.g., the whole kappa light chain constant region and the Cmdomain of the heavy chain constant region, with or without the heavychain hinge domain. Thus, the genes encoding the antibody segments mayoccur in any order on a single plasmid, or may be expressed separatelyfrom separate plasmids. For example, in another embodiment of theinvention, the V_(L) gene and any light chain constant region will be onone plasmid, while the V_(H) gene and any heavy chain constant regionwill be on a second plasmid. In either case, the V_(L) and/or V_(H)genes may be preceded by a signal sequence that directs the secretion ofthe recombinant fusion protein from the cell. See, e.g., U.S. Pat. Nos.6,147,203, 6,074,644, 6,051,405 which are incorporated herein byreference in their entirety.

In another embodiment, the invention provides for single chainantibodies, in which the antibody comprises the V_(L) or V_(H) regionsalone, rather than as components of Fv fragments. See, e.g., U.S. Pat.No. 6,074,644, which is incorporated herein by reference in itsentirety.

An antibody can also be conjugated to a second antibody to form anantibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety. Ina specific embodiment, two or more antibodies are cross-linked to eachto produce a bispecific or multispecific antibody.

In one embodiment, an antibody of the invention is not only found to bespecific for IL3Rα but also binds with very high affinity, based onbinding assays using IL3Rα-expressing cell lines. In another embodiment,an antibody of the invention is not only found to be specific for IL3Rαbut also binds an epitope that overlaps with that bound by the 26292antibody. In other embodiments, an antibody of the invention is not onlyfound to be specific for IL3Rα but also binds an epitope that overlapswith that bound by the 32703, 32701, or 32716 antibody.

The present invention is based, in part, upon the discovery that eventhough a particular anti-IL3Rα antibody might bind to IL3Rα specificallyand with high affinity, that does not guarantee that it will constitutean effective immunotoxin when linked to a cytotoxic agent. In oneembodiment, antibodies with high affinity and specificity are chosen forconjugation to a cytotoxic agent, and they are then assessed for theirability to selectively and potently kill IL3Rα-expressing cells. In aspecific embodiment, the IL3Rα antibody binds an epitope that overlapsthat of an antibody known to cause specific cytotoxicity toIL3Rα-expressing cells. For example, in one embodiment, an IL3Rαantibody is tested for epitope overlap with the anti-IL3Rα antibody26292, as assessed by the following competition assay: Cells that bearIL3Rα (e.g., TF-1, Molm-13, or Molm-14) are incubated with an excess ofthe 26292 antibody for 1 hour at 4° C., followed by incubation for 1hour with an excess of the second IL3Rα antibody. If the two antibodiesbind to an overlapping epitope, the amount bound by the combination willbe close to that of either single antibody, whereas if the twoantibodies bind to different epitopes, the signal from the combinationwould be close to the sum of two individual values. Bound antibody isdetected with the fluorescently-labeled secondary antibody R-PE(phycoerythrin)-labeled goat F(ab′)₂ anti-rabbit IgG (BioSource) orother appropriate specific fluorescently-labeled probe. In otherembodiments, an IL3Rα antibody is tested for epitope overlap with theanti-IL3Rα antibody 32703, 32701, or 32716.

In one embodiment, an IL3Rα antibody binds to the same epitope as the26292, 32703, 32701, or 32716 antibody as assessed by a competitionassay known to one of skill in the art or described herein. In anotheralternative embodiment, the IL3Rα antibody does not bind to the sameepitope as the 26292, 32703, 32701, or 32716 antibody, as assessed by acompetition assay known to one of skill in the art or described herein.

In another embodiment of this invention, the anti-IL3Rα antibodycompetes with IL-3 for binding to IL3Rα or a fragment thereof (e.g., theextracellular domain of the IL3Rα or a fragment thereof). In a specificembodiment, the anti-IL3Rα antibody competes with IL-3 for binding toIL3Rα-expressing cells. In some embodiments, an antibody of theinvention prevents or reduces IL3R signal transduction. In specificembodiments, an antibody of the invention binds to the IL3Rα and theantibody-IL3Rαcomplex is internalized.

The present invention provides antibodies that exhibit a highassociation rate (k_(on)) value in an assay known to one of skill in theart or described herein, e.g., a plasmon resonance assay. See, e.g.,U.S. Application Publication No. 20020098189 (which is incorporatedherein by reference in its entirety) for methods for producing andidentifying antibodies with a high k_(on), value. In a specificembodiment, an antibody of the present invention has an association rateconstant or k_(on), rate (antibody (Ab)+antigen (Ag)^(k) ^(on) →Ab—Ag)of at least 2×10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻¹s⁻¹, at least 10⁶ M⁻¹s⁻¹, atleast 5×10⁶ M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹, at least 5×10⁷ M⁻¹s⁻¹, or atleast 10⁸ M⁻¹s⁻¹.

The present invention that have a low dissociation rate (k_(off)) in anassay known to one of skill in the art or described herein, e.g., aplasmon resonance assay. In a specific embodiment, an antibody of theinvention has a k_(off) rate (antibody (Ab)+antigen (Ag)^(K) ^(off)→Ab—Ag) of less than 10⁻³s⁻¹, less than 5×10⁻³s⁻¹, less than 10's⁻¹,less than 5×10⁻⁴s⁻¹, less than 10⁻⁵s⁻¹, less than 5×10⁻⁵s⁻¹, less than10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷s⁻¹, less than 5×10⁻⁷s⁻¹,less than 10⁻⁸s⁻¹, less than 5×10⁻⁸s⁻¹, less than 10⁻⁹ s⁻¹, less than5×10⁻⁹s⁻¹, or less than 10⁻¹⁰ s⁻¹.

The present invention provides antibodies that bind to the IL3Rα chainwith high affinity. In a specific embodiment, an antibody of theinvention has an affinity constant or K_(a) (k_(on)/k_(off)) of at least5×10⁶ M⁻¹, at least 10⁷ M⁻¹, at least 5×10⁷ M⁻¹, at least 10⁸ M⁻¹ atleast 5×10⁸ M⁻¹ at least 10⁹ M⁻¹, at least 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹,at least 5×10¹⁰ M⁻¹, at least 10¹¹ M⁻¹ at least 5×10¹¹ M⁻¹, at least10¹² M⁻¹ at least 5×10¹² M⁻¹, at least 10¹³ M⁻¹, at least 5×10¹³ M⁻¹, atleast 10¹⁴ M⁻¹, at least 5×10¹⁴ M⁻¹, at least 10¹⁵ M⁻¹, or at least5×10¹⁵ M⁻¹. In another embodiment, the antibodies of the invention has aK_(a) of between 5×10⁶ M⁻¹ to 5×10¹⁵ M⁻¹, 1×10⁷ M⁻¹ to 5×10¹² M⁻¹, or5×10⁷ M⁻¹ to 5×10¹⁰ M⁻¹.

In another embodiment, an antibody has a dissociation constant or K_(a)(k_(off)/k_(on)) of less than 5×10⁻³ M, less than 10⁻⁴ M, less than5×10⁻⁴ M, less than 10⁻⁵ M, less than 5×10⁻⁵ M, less than 10⁻⁶ M, lessthan 5×10⁻⁶ M, less than 10⁻⁷ M, less than 5×10⁻⁷ M, less than 10⁻⁸ M,less than 5×10⁻⁸ M, less than 10⁻⁹ M, less than 5×10⁻⁹ M, less than10⁻¹⁰ M, less than 5×10⁻¹⁰ M, less than 10⁻¹¹ M, less than 5×10⁻¹¹ M,less than 10⁻¹² M, less than 5×10⁻¹² M, less than 10⁻¹³ M, less than5×10⁻¹³ M, less than 10⁻¹⁴ M, less than 5×10⁻¹⁴ M, less than 10⁻¹⁵ M, orless than 5×10⁻¹⁵ M. In another embodiment, the antibodies of theinvention have a dissociation constant (K_(d)) of less than 4000 pM,less than 3700 pM, less than 3500 pM, less than 3000 pM, less than 2750pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less than1000 pM, less than 750 pM, less than 500 pM, less than 250 pM, less than200 pM, less than 150 pM, less than 100 pM, or less than 75 pM asassessed using an assay described herein or known to one of skill in theart (e.g., a BIAcore assay). In a specific embodiment, the antibodiesused in accordance with the methods of the invention bind to the IL3Rαantigen and have a dissociation constant (K_(D)) of between 25 to 4000pM, 25 to 3750 pM, 25 to 3500 pM, or 25 to 3000 pM as assessed using anassay described herein or known to one of skill in the art (e.g., aBIAcore assay).

The present invention provides antibodies that have a half-maximalinhibitory concentration (IC₅₀) of less than 50 ng/ml, less than 45ng/ml, less than 40 nM, less than 35 nM, or less than 30 nM as assessedby an assay known to one of skill in the art or described herein. In aspecific embodiment, the antibodies of the invention have an IC₅₀ ofbetween 25 ng/ml to 75 ng/ml, 25 ng/ml to 50 ng/ml, or 25 ng/ml to 40ng/ml.

In a specific embodiment, an antibody of the present invention is themonoclonal antibody 26292. In another embodiment, an antibody of thepresent invention is the scFv 26929. FIG. 1 and Table 1 provides theamino acid sequences for the V_(H) and V_(L) domains of the 26292monoclonal or scFv antibody.

In one embodiment, an antibody of the present invention is themonoclonal antibody 32703. In another embodiment, an antibody of thepresent invention is the scFv antibody 32703. FIG. 2 and Table 1provides the amino acid sequences for the V_(H) and V_(L) domains of themonoclonal or scFv antibody 32703.

In one embodiment, an antibody of the present invention is themonoclonal antibody 32701. In another embodiment, an antibody of thepresent invention is the scFv antibody 32701. FIG. 3 and Table 1 providethe amino acid sequences for the variable heavy (V_(H)) and variablelight (V_(L)) domains of the monoclonal or scFv antibody 32701. The32703 monoclonal antibody is available from R&D Systems, Inc.

In one embodiment, an antibody of the present invention is themonoclonal antibody 32716. In another embodiment, an antibody of thepresent invention is the scFv antibody 32701. FIG. 4 and Table 1 providethe amino acid sequences for the variable heavy (V_(H)) and variablelight (V_(L)) domains of the monoclonal or scFv antibody 32716.

TABLE 1 Amino Acid Sequences of the 26292, 32703, 32701, and 32716Antibodies Antibody 26292 32703 32701 32716 V_(H) domain SEQ ID NO: 2SEQ ID NO: SEQ ID NO: SEQ ID NO: 12 22 32 V_(H) CDR1 SEQ ID NO: 3 SEQ IDNO: SEQ ID NO: SEQ ID NO: 13 23 33 V_(H) CDR1 SEQ ID NO: 4 SEQ ID NO:SEQ ID NO: SEQ ID NO: 14 24 34 V_(H) CDR3 SEQ ID NO: 5 SEQ ID NO: SEQ IDNO: SEQ ID NO: 15 25 35 V_(L) domain SEQ ID NO: 7 SEQ ID NO: SEQ ID NO:SEQ ID NO: 17 27 37 V_(L) CDR1 SEQ ID NO: 8 SEQ ID NO: SEQ ID NO: SEQ IDNO: 18 28 38 V_(L) CDR2 SEQ ID NO: 9 SEQ ID NO: SEQ ID NO: SEQ ID NO: 1929 39 V_(L) CDR3 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 10 20 30 40

In a specific embodiment, the present invention provides antibodies thatbind to the IL3Rα chain, said antibodies comprising a V_(H) domainand/or V_(L) domain having the amino acid sequence of a V_(H) domainand/or V_(L) domain of 26292, 32703, 32701, or 32716. In someembodiments, the antibodies comprise a human constant region known toone of skill in the art.

In one embodiment, the present invention provides antibodies that bindto the IL3Rα chain, said antibodies comprising one or more CDRs havingthe amino acid sequence of one or more CDRs of the monoclonal or scFvantibody 26292. In another embodiment, the present invention providesantibodies that bind to the IL3Rα chain, said antibodies comprising acombination of V_(H) CDRs and/or V_(L) CDRs having the amino acidsequence of V_(H) CDRs and/or V_(L) CDRs of the monoclonal or scFvantibody 26292.

In one embodiment, the present invention provides antibodies that bindto the IL3Rα chain, said antibodies comprising one or more CDRs havingthe amino acid sequence of one or more CDRs of the monoclonal or scFvantibody 32703. In another embodiment, the present invention providesantibodies that bind to the IL3Rα chain, said antibodies comprising acombination of V_(H) CDRs and/or V_(L) CDRs having the amino acidsequence of V_(H) CDRs and/or V_(L) CDRs of the monoclonal or scFvantibody 32703.

In one embodiment, the present invention provides antibodies that bindto the IL3Rα chain, said antibodies comprising one or more CDRs havingthe amino acid sequence of one or more CDRs of the monoclonal or scFvantibody 32701. In another embodiment, the present invention providesantibodies that bind to the IL3Rα chain, said antibodies comprising acombination of V_(H) CDRs and/or V_(L) CDRs having the amino acidsequence of V_(H) CDRs and/or V_(L) CDRs of the monoclonal or scFvantibody 32701.

In one embodiment, the present invention provides antibodies that bindto the IL3Rα chain, said antibodies comprising one or more CDRs havingthe amino acid sequence of one or more CDRs of the monoclonal or scFvantibody 32716. In another embodiment, the present invention providesantibodies that bind to the IL3Rα chain, said antibodies comprising acombination of V_(H) CDRs and/or V_(L) CDRs having the amino acidsequence of V_(H) CDRs and/or V_(L) CDRs of the monoclonal or scFvantibody 32716.

The present invention provides antibodies that bind to the IL3Rα chain,said antibodies comprising one or more VH CDRs and one or more VL CDRslisted in Table 1, supra. In particular, the invention provides anantibody that binds to the IL3Rα chain, said antibody comprising (oralternatively, consisting of) a VH CDR1 of the 26292, 32703, 32701 or32719 antibody and a VL CDR1 of the 26292, 32703, 32701 or 32719antibody; a VH CDR1 of the 26292, 32703, 32701 or 32719 antibody and aVL CDR2 of the 26292, 32703, 32701 or 32719 antibody; a VH CDR1 of the26292, 32703, 32701 or 32719 antibody and a VL CDR3 of the 26292, 32703,32701 or 32719 antibody; a VH CDR2 of the 26292, 32703, 32701 or 32719antibody and a VL CDR1 of the 26292, 32703, 32701 or 32719 antibody; VHCDR2 of the 26292, 32703, 32701 or 32719 antibody and VL CDR2 of the26292, 32703, 32701 or 32719 antibody; a VH CDR2 of the 26292, 32703,32701 or 32719 antibody and a VL CDR3 of the 26292, 32703, 32701 or32719 antibody; a VH CDR3 of the 26292, 32703, 32701 or 32719 antibodyand a VH CDR1 of the 26292, 32703, 32701 or 32719 antibody; a VH CDR3 ofthe 26292, 32703, 32701 or 32719 antibody and a VL CDR2 of the 26292,32703, 32701 or 32719 antibody; a VH CDR3 of the 26292, 32703, 32701 or32719 antibody and a VL CDR3 of the 26292, 32703, 32701 or 32719antibody; a VH1 CDR1, a VH CDR2 and a VL CDR1 of the 26292, 32703, 32701or 32719 antibody; a VH CDR1, a VH CDR2 and a VL CDR2 of the 26292,32703, 32701 or 32719 antibody; a VH CDR1, a VH CDR2 and a VL CDR3 ofthe 26292, 32703, 32701 or 32719 antibody; a VH CDR2, a VH CDR3 and a VLCDR1, a VH CDR2, a VH CDR3 and a VL CDR2 of the 26292, 32703, 32701 or32719 antibody; a VH CDR2, a VH CDR2 and a VL CDR3 of the 26292, 32703,32701 or 32719 antibody; a VH CDR1, a VL CDR1 and a VL CDR2 of the26292, 32703, 32701 or 32719 antibody; a VH CDR1, a VL CDR1 and a VLCDR3 of the 26292, 32703, 32701 or 32719 antibody; a VH CDR2, a VL CDR1and a VL CDR2 of the 26292, 32703, 32701 or 32719 antibody; a VH CDR2, aVL CDR1 and a VL CDR3 of the 26292, 32703, 32701 or 32719 antibody; a VHCDR3, a VL CDR1 and a VL CDR2 of the 26292, 32703, 32701 or 32719antibody; a VH CDR3, a VL CDR1 and a VL CDR3 of the 26292, 32703, 32701or 32719 antibody; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR1 of the26292, 32703, 32701 or 32719 antibody; a VH CDR1, a VH CDR2, a VH CDR3and a VL CDR2 of the 26292, 32703, 32701 or 32719 antibody; a VH CDR1, aVH CDR2, a VH CDR3 and a VL CDR3 of the 26292, 32703, 32701 or 32719antibody; a VH CDR1, a VH CDR2, a VL CDR1 and a VL CDR2 of the 26292,32703, 32701 or 32719 antibody; a VH CDR1, a VH CDR2, a VL CDR1 and a VLCDR3 of the 26292, 32703, 32701 or 32719 antibody; a VH CDR1, a VH CDR3,a VL CDR1 and a VL CDR2 of the 26292, 32703, 32701 or 32719 antibody; aVH CDR1, a VH CDR3, a VL CDR1 and a VL CDR3 of the 26292, 32703, 32701or 32719 antibody; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2 of the26292, 32703, 32701 or 32719 antibody; a VH CDR2, a VH CDR3, a VL CDR1and a VL CDR3 of the 26292, 32703, 32701 or 32719 antibody; a VH CDR2, aVH CDR3, a VL CDR2 and a VL CDR3 of the 26292, 32703, 32701 or 32719antibody; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2 ofthe 26292, 32703, 32701 or 32719 antibody; a VH CDR1, a VH CDR2, a VHCDR3, a VL CDR1 and a VL CDR3 of the 26292, 32703, 32701 or 32719antibody; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VL CDR3 ofthe 26292, 32703, 32701 or 32719 antibody; a VH CDR1, a VH CDR3, a VLCDR1, a VL CDR2, and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR1, a VLCDR2, and a VL CDR3 of the 26292, 32703, 32701 or 32719 antibody; or anycombination thereof of the VH CDRs and VL CDRs listed in Table 1, supra.

The present invention provides antibodies that bind to the IL3Rα chain,said antibodies comprising a V_(H) domain disclosed herein combined witha V_(L) domain disclosed herein, or other V_(L) domain. The presentinvention also provides antibodies that bind to the IL3Rα chain, saidantibodies comprising a V_(L) domain disclosed herein combined with aV_(H) domain disclosed herein, or other V_(H) domain.

The present invention provides for nucleic acid molecules, generallyisolated, comprising a nucleotide sequence(s) encoding an antibody ofthe invention. The nucleic acid sequences of the V_(H) domains, theV_(L) domains, V_(H) CDRs and V_(L) CDRs are provided in FIGS. 1A, 1C,2A, 2C, 3A, 3C, 4A and 4C. The invention encompasses any nucleic acidsequence that encodes an antibody of the invention. In a specificembodiment, an isolated nucleic acid molecule(s) of the inventionencodes the 26292 antibody or an antigen-binding fragment thereof. Inanother embodiment, an isolated nucleic acid molecule(s) of theinvention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(H) domain having an amino acid sequence of theV_(H) domain of the 26292 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the invention encodes an antibody that bindsto the IL3Rα chain, the antibody comprising a V_(L) domain having anamino acid sequence of the V_(L) domain the 26292 antibody. In anotherembodiment, isolated nucleic acid molecules of the invention encode anantibody that binds to the IL3Rα chain, the antibody comprising a V_(H)domain and a V_(L) domain having an amino acid sequence of the V_(H)domain and V_(L) domain of the 26292 antibody.

In a another embodiment, an isolated nucleic acid molecule(s) of theinvention encodes the 32703 antibody or an antigen-binding fragmentthereof. In another embodiment, an isolated nucleic acid molecule(s) ofthe invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(H) domain having an amino acid sequence of theV_(H) domain of the 32703 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the invention encodes an antibody that bindsto the IL3Rα chain, the antibody comprising a V_(L) domain having anamino acid sequence of the V_(L) domain the 32703 antibody. In anotherembodiment, isolated nucleic acid molecules of the invention encode anantibody that binds to the IL3Rα chain, the antibody comprising a V_(H)domain and a V_(L) domain having an amino acid sequence of the V_(H)domain and V_(L) domain of the 32703 antibody.

In a another embodiment, an isolated nucleic acid molecule(s) of theinvention encodes the 32701 antibody or an antigen-binding fragmentthereof. In another embodiment, an isolated nucleic acid molecule(s) ofthe invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(H) domain having an amino acid sequence of theV_(H) domain of the 32701 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the invention encodes an antibody that bindsto the IL3Rα chain, the antibody comprising a V_(L) domain having anamino acid sequence of the V_(L) domain the 32701 antibody. In anotherembodiment, isolated nucleic acid molecules of the invention encode anantibody that binds to the IL3Rα chain, the antibody comprising a V_(H)domain and a V_(L) domain having an amino acid sequence of the V_(H)domain and V_(L) domain of the 32701 antibody.

In a another embodiment, an isolated nucleic acid molecule(s) of theinvention encodes the 32716 antibody or an antigen-binding fragmentthereof. In another embodiment, an isolated nucleic acid molecule(s) ofthe invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(H) domain having an amino acid sequence of theV_(H) domain of the 32716 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the invention encodes an antibody that bindsto the IL3Rα chain, the antibody comprising a V_(L) domain having anamino acid sequence of the V_(L) domain the 32716 antibody. In anotherembodiment, isolated nucleic acid molecules of the invention encode anantibody that binds to the IL3Rα chain, the antibody comprising a V_(H)domain and a V_(L) domain having an amino acid sequence of the V_(H)domain and V_(L) domain of the 32716 antibody.

In another embodiment, an isolated nucleic acid molecule(s) of theinvention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(H) CDR1 having an amino acid sequence of theV_(H) CDR1 of the 26292 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the invention encodes an antibody that bindsto the IL3Rα chain, the antibody comprising a V_(H) CDR2 having an aminoacid sequence of the V_(H) CDR2 of the 26292 antibody. In yet anotherembodiment, an isolated nucleic acid molecule(s) of the inventionencodes an antibody that binds to the IL3Rα chain, the antibodycomprising a V_(H) CDR3 having an amino acid sequence of the V_(H) CDR3of the 26292 antibody.

In another embodiment, an isolated nucleic acid molecule(s) of theinvention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(H) CDR1 having an amino acid sequence of theV_(H) CDR1 of the 32703 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the invention encodes an antibody that bindsto the IL3Rα chain, the antibody comprising a V_(H) CDR2 having an aminoacid sequence of the V_(H) CDR2 of the 32703 antibody. In yet anotherembodiment, an isolated nucleic acid molecule(s) of the inventionencodes an antibody that binds to the IL3Rα chain, the antibodycomprising a V_(H) CDR3 having an amino acid sequence of the V_(H) CDR3of the 32703 antibody.

In another embodiment, an isolated nucleic acid molecule(s) of theinvention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(H) CDR1 having an amino acid sequence of theV_(H) CDR1 of the 32701 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the invention encodes an antibody that bindsto the IL3Rα chain, the antibody comprising a V_(H) CDR2 having an aminoacid sequence of the V_(H) CDR2 of the 32701 antibody. In yet anotherembodiment, an isolated nucleic acid molecule(s) of the inventionencodes an antibody that binds to the IL3Rα chain, the antibodycomprising a V_(H) CDR3 having an amino acid sequence of the V_(H) CDR3of the 32701 antibody.

In another embodiment, an isolated nucleic acid molecule(s) of theinvention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(H) CDR1 having an amino acid sequence of theV_(H) CDR1 of the 32716 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the invention encodes an antibody that bindsto the IL3Rα chain, the antibody comprising a V_(H) CDR2 having an aminoacid sequence of the V_(H) CDR2 of the 32716 antibody. In yet anotherembodiment, an isolated nucleic acid molecule(s) of the inventionencodes an antibody that binds to the IL3Rα chain, the antibodycomprising a V_(H) CDR3 having an amino acid sequence of the V_(H) CDR3of the 32716 antibody.

In another embodiment, an isolated nucleic acid molecule(s) of thepresent invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(L) CDR1 having an amino acid sequence of theV_(L) CDR1 of the 26292 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the present invention encodes an antibodythat binds to the IL3Rα chain, the antibody comprising a V_(L) CDR2having an amino acid sequence of the V_(L) CDR2 of the 26292 antibody.In yet another embodiment, an isolated nucleic acid molecule(s) of thepresent invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(L) CDR3 having an amino acid sequence of theV_(L) CDR3 of the 26292 antibody.

In another embodiment, an isolated nucleic acid molecule(s) of thepresent invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(L) CDR1 having an amino acid sequence of theV_(L) CDR1 of the 32703 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the present invention encodes an antibodythat binds to the IL3Rα chain, the antibody comprising a V_(L) CDR2having an amino acid sequence of the V_(L) CDR2 of the 32703 antibody.In yet another embodiment, an isolated nucleic acid molecule(s) of thepresent invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(L) CDR3 having an amino acid sequence of theV_(L) CDR3 of the 32703 antibody.

In another embodiment, an isolated nucleic acid molecule(s) of thepresent invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(L) CDR1 having an amino acid sequence of theV_(L) CDR1 of the 32701 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the present invention encodes an antibodythat binds to the IL3Rα chain, the antibody comprising a V_(L) CDR2having an amino acid sequence of the V_(L) CDR2 of the 32701 antibody.In yet another embodiment, an isolated nucleic acid molecule(s) of thepresent invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(L) CDR3 having an amino acid sequence of theV_(L) CDR3 of the 32701 antibody.

In another embodiment, an isolated nucleic acid molecule(s) of thepresent invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(L) CDR1 having an amino acid sequence of theV_(L) CDR1 of the 32716 antibody. In another embodiment, an isolatednucleic acid molecule(s) of the present invention encodes an antibodythat binds to the IL3Rα chain, the antibody comprising a V_(L) CDR2having an amino acid sequence of the V_(L) CDR2 of the 32716 antibody.In yet another embodiment, an isolated nucleic acid molecule(s) of thepresent invention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a V_(L) CDR3 having an amino acid sequence of theV_(L) CDR3 of the 32716 antibody.

In another embodiment, a nucleic acid molecule(s) of the inventionencodes an antibody that binds to the IL3Rα chain, the antibodycomprising a VH CDR1, a VL CDR1, a VH CDR2, a VL CDR2, a VH CDR3, a VLCDR3, or any combination thereof having an amino acid sequence the 26292antibody. In another embodiment, a nucleic acid molecule(s) of theinvention encodes an antibody that binds to the IL3Rα chain, theantibody comprising a VH CDR1, a VL CDR1, a VH CDR2, a VL CDR2, a VHCDR3, a VL CDR3, or any combination thereof having an amino acidsequence the 32703 antibody. In another embodiment, a nucleic acidmolecule(s) of the invention encodes an antibody that binds to the IL3Rαchain, the antibody comprising a VH CDR1, a VL CDR1, a VH CDR2, a VLCDR2, a VH CDR3, a VL CDR3, or any combination thereof having an aminoacid sequence the 32701 antibody. In another embodiment, a nucleic acidmolecule(s) of the invention encodes an antibody that binds to the IL3Rαchain, the antibody comprising a VH CDR1, a VL CDR1, a VH CDR2, a VLCDR2, a VH CDR3, a VL CDR3, or any combination thereof having an aminoacid sequence the 32716 antibody.

In one embodiment, the invention provides for a nucleic acid comprisinga nucleotide sequence encoding an antibody comprising a V_(H) region ofan Fv fragment linked to the nucleotides that encode the V_(L) regionthrough nucleotides that encode a peptide linker. Alternatively, or incombination, the encoded V_(H) region contains cysteine residues thatform disulfide linkages with a cysteine-containing V_(L) region of an Fvfragment. In another embodiment, this invention provides for nucleicacids comprising nucleotide sequences encoding any of the antibodiesdescribed herein, in which the antibody comprises the V_(L) or V_(H)region alone. See, e.g., U.S. Pat. No. 6,074,644, which is referencedherein in its entirety.

Those skilled in the art will realize that additional modifications,deletions, insertions and the like may be made to the IL3Rα antibody.Especially, deletions or other changes may be made to the antibody inorder to increase stability, affinity, specificity, or, when combinedwith the cytotoxic agent, cytotoxicity or other impairment toIL3Rα-expressing cells and/or to decrease non-specific cytotoxicity orimpairment toward cells that lack IL3Rα antigens. Typical modificationsinclude, but are not limited to, introduction of an upstream methioninefor transcription initiation, mutation of residues to cysteine in V_(H)or V_(L) regions for the creation of disulfide linkages, etc. All suchconstructions may be made by methods of genetic engineering well knownto those skilled in the art. Fragments, analogs, and derivatives ofIL3Rα antibodies can be useful in the present invention provided thatwhen fused to the cytotoxic agent portion of the conjugate, suchfragments, analogs, and derivatives maintain the ability to bind nativeIL3Rα expressed on the surface of a cell. Preferably, the bindingkinetics of the fragments, analogs, or derivatives remain the same orvary by no more than 25% (preferably, no more than 15%, 10% or 5%) asdetermined by an assays described herein.

To improve or alter the characteristics of IL3Rα antibodies, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins” including single or multiple amino acid substitutions,deletions, additions, or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions. For instance, for many proteins, it is known inthe art that one or more amino acids may be deleted from the aminoterminus or carboxyl terminus without substantial loss of biologicalfunction.

Antibodies may be altered by random mutagenesis by error-prone PCR,random nucleotide insertion or other methods prior to recombination.

The present invention also provides antibodies that bind to the IL3Rαchain, the antibodies comprising derivatives of the V_(H) domains, V_(H)CDRs, V_(L) domains, and V_(L) CDRs described herein. Standardtechniques known to those of skill in the art can be used to introducemutations in the nucleotide sequence encoding a molecule of theinvention, including, for example, site directed mutagenesis and PCRmediated mutagenesis which results in amino acid substitutions.Preferably, the derivatives include less than 25 amino acidsubstitutions, less than 20 amino acid substitutions, less than 15 aminoacid substitutions, less than 10 amino acid substitutions, less than 5amino acid substitutions, less than 4 amino acid substitutions, lessthan 3 amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original molecule. In a preferred embodiment, thederivatives have conservative amino acid substitutions are made at oneor more predicted non-essential amino acid residues. A “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a side chain with a similarcharge. Families of amino acid residues having side chains with similarcharges have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), non-polar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan), betabranched side chains (e.g., threonine, valine, isoleucine) and aromaticside chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).Alternatively, mutations can be introduced randomly along all or part ofthe coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for biological activity to identifymutants that retain activity. Following mutagenesis, the encoded proteincan be expressed and the activity of the protein can be determined.

The present invention provides antibodies that bind to the IL3Rα chain,said antibodies comprising the amino acid sequence of the V_(H) and/orV_(L) domain or an antigen-binding fragment thereof of the 26292antibody with one, two, three, four, five, six or more amino acidresidue substitutions in the V_(H) and/or V_(L) domain orantigen-binding fragment. The present invention also provides antibodiesthat bind to the IL3Rα chain, said antibodies comprising the amino acidsequence of the V_(H) and/or V_(L) domain or an antigen-binding fragmentthereof of the 26292 antibody with one, two, three, four, five, six ormore amino acid residue substitutions in one or more V_(H) CDRs and/orone or more V_(L) CDRs. In a specific embodiment, the amino acidsubstitution(s) is a conservative amino acid substitution(s).

In an embodiment, an antibody that binds to the IL3Rα chain comprisesthe amino acid sequence of the V_(H) and/or V_(L) domain or anantigen-binding fragment thereof of the 32703 antibody with one, two,three, four, five, six or more amino acid residue substitutions in theV_(H) and/or V_(L) domain or antigen-binding fragment. The presentinvention also provides antibodies that bind to the IL3Rα chain, saidantibodies comprising the amino acid sequence of the V_(H) and/or V_(L)domain or an antigen-binding fragment thereof of the 32703 antibody withone, two, three, four, five, six or more amino acid residuesubstitutions in one or more V_(H) CDRs and/or one or more V_(L) CDRs.In a specific embodiment, the amino acid substitution(s) is aconservative amino acid substitution(s).

In another embodiment, an antibody that binds to the IL3Rα chaincomprises the amino acid sequence of the V_(H) and/or V_(L) domain or anantigen-binding fragment thereof of the 32701 antibody with one, two,three, four, five, six or more amino acid residue substitutions in theV_(H) and/or V_(L) domain or antigen-binding fragment. The presentinvention also provides antibodies that bind to the IL3Rα chain, saidantibodies comprising the amino acid sequence of the V_(H) and/or V_(L)domain or an antigen-binding fragment thereof of the 32701 antibody withone, two, three, four, five, six or more amino acid residuesubstitutions in one or more V_(H) CDRs and/or one or more V_(L) CDRs.In a specific embodiment, the amino acid substitution(s) is aconservative amino acid substitution(s).

In another embodiment, an antibody that binds to the IL3Rα chaincomprises the amino acid sequence of the V_(H) and/or V_(L) domain or anantigen-binding fragment thereof of the 32716 antibody with one, two,three, four, five, six or more amino acid residue substitutions in theV_(H) and/or V_(L) domain or antigen-binding fragment. The presentinvention also provides antibodies that bind to the IL3Rα chain, saidantibodies comprising the amino acid sequence of the V_(H) and/or V_(L)domain or an antigen-binding fragment thereof of the 32716 antibody withone, two, three, four, five, six or more amino acid residuesubstitutions in one or more V_(H) CDRs and/or one or more V_(L) CDRs.In a specific embodiment, the amino acid substitution(s) is aconservative amino acid substitution(s).

The present invention also provides antibodies that bind to the IL3Rαchain, said antibodies comprising the amino acid sequence of thevariable heavy domain and/or variable light domain or an antigen-bindingfragment thereof of the 26292 antibody with one or more amino acidresidue substitutions in one or more V_(H) frameworks and/or one or moreV_(L) frameworks. In one embodiment, the antibody that bind to the IL3Rαchain comprises the amino acid sequence of the variable heavy domainand/or variable light domain or an antigen-binding fragment thereof ofthe 32703 antibody with one or more amino acid residue substitutions inone or more V_(H) frameworks and/or one or more V_(L) frameworks. In oneembodiment, the antibody that bind to the IL3Rα chain comprises theamino acid sequence of the variable heavy domain and/or variable lightdomain or an antigen-binding fragment thereof of the 32701 antibody withone or more amino acid residue substitutions in one or more V_(H)frameworks and/or one or more V_(L) frameworks. In one embodiment, theantibody that bind to the IL3Rα chain comprises the amino acid sequenceof the variable heavy domain and/or variable light domain or anantigen-binding fragment thereof of the 32716 antibody with one or moreamino acid residue substitutions in one or more V_(H) frameworks and/orone or more V_(L) frameworks. The antibody generated by introducingsubstitutions in the V_(H) domain, V_(H) CDRs, V_(L) domain, V_(L) CDRs,and/or frameworks of the 26292, 32703, 32701, or 32716 antibodies can betested in vitro and/or in vivo, for example, for its ability to bind toan IL3Rα antigen, or for its ability to, when conjugated to a cytotoxin,specifically kill or otherwise impair cells that express IL3Rα.

In a specific embodiment, an antibody that binds to the IL3Rα chaincomprises an amino acid sequence encoded by a nucleotide sequence thathybridizes to the nucleotide sequence(s) encoding the 26292, 32703,32701, or 32716 antibody or an antigen-binding fragment thereof understringent conditions, e.g., hybridization to filter-bound DNA in 6×sodium chloride/sodium citrate (SSC) at about 45 followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C., under highly stringentconditions, e.g., hybridization to filter-bound nucleic acid in 6×SSC atabout 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about68° C., or under other stringent hybridization conditions that are knownto those of skill in the art (see, for example, Ausubel et al., eds.,1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3).

In another embodiment, an antibody that binds to the IL3Rα chaincomprises an amino acid sequence of a V_(H) domain and/or an amino acidsequence a V_(L) domain encoded by a nucleotide sequence that hybridizesto the nucleotide sequence encoding the V_(H) and/or V_(L) domains ofthe 26292, 32703, 32701, or 32716 antibody under stringent conditions,e.g., hybridization to filter-bound DNA in 6×SSC at about 45° C.followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C.,under highly stringent conditions, e.g., hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C., or under other stringent hybridizationconditions that are known to those of skill in the art (see, forexample, Ausubel, et al., eds., 1989, Current Protocols in MolecularBiology, Vol. I, Green Publishing Associates, Inc. and John Wiley &Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).

In another embodiment, an antibody that binds to the IL3Rα chaincomprises an amino acid sequence of a V_(H) CDR and/or an amino acidsequence of a V_(L) CDRs encoded by a nucleotide sequence thathybridizes to the nucleotide sequence encoding the V_(H) CDRs and/orV_(L) CDRs of the 26292, 32703, 32701, or 32716 antibody under stringentconditions, e.g., hybridization to filter-bound DNA in 6×SSC at about45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about50-65° C., under highly stringent conditions, e.g., hybridization tofilter-bound nucleic acid in 6×SSC at about 45° C. followed by one ormore washes in 0.1×SSC/0.2% SDS at about 68° C., or under otherstringent hybridization conditions that are known to those of skill inthe art.

In a specific embodiment, an antibody that binds to the IL3Rα chaincomprises an amino acid sequence that is at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical to the amino acid sequence of the26292, 32703, 32701 or 32716 antibody or an antigen-binding fragmentthereof.

In another embodiment, an antibody that binds to the IL3Rα chaincomprises an amino acid sequence of a V_(H) domain and/or an amino acidsequence of a V_(L) domain that is at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 99% identical to the V_(H) domain and/or V_(L) domainof the 26292, 32703, 32701 or 32716 antibody. In another embodiment, anantibody that binds to the IL3Rα chain comprises an amino acid sequenceof one or more V_(H) CDRs and/or an amino acid sequence of one or moreV_(L) CDRs that are at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least99% identical to one or more of the V_(H) CDRs and/or one or more of theV_(L) CDRs of the 26292, 32703, 32701, or 32716 antibody.

The present invention also encompasses antibodies that compete with the26292, 32703, 32701, or 32716 antibody for binding to the IL3Rα chain(preferably, human IL3Rα chain). The invention also encompassesantibodies that compete with an antibody comprising the VH domain and VLdomain of the 26292, 32703, 32701 or 32716 antibody or an antibodycomprising the VH CDRs and VL CDRs of the 26292, 32703, 32701 or 32716antibody for binding to the IL3Rα chain (preferably, the human IL3Rαchain). The present invention also encompasses polypeptides, proteins,and peptides comprising V_(L) domains and/or V_(H) domains that competewith a V_(L) domain and/or a V_(H) domain of the 26292, 32703, 32701, or32716 antibody for binding to the IL3Rα chain (preferably, the humanIL3Rα chain). Further, the present invention encompasses polypeptides,proteins, and peptides comprising V_(L) CDRs and/or V_(H) CDRs thatcompete with a V_(L) CDR and/or V_(H) CDR of the 26292, 32703, 32701, or32716 antibody for binding to the IL3Rα chain (preferably, the humanIL3Rα chain).

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not adversely affect binding to theantigen or, in the case of antibody conjugates of the invention,adversely affect its ability to bind to the IL3Rα chain and adverselyaffect its ability to specifically kill or impair cells that express theIL3Rα chain. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

The present invention also provides antibodies that bind to the IL3Rαchain that comprise a framework region known to those of skill in theart (e.g., a human or non-human fragment). The framework region may benaturally occurring or consensus framework regions. In one embodiment,the framework region of an antibody of the invention is human (see,e.g., Clothia et al., 1998, J. Mol. Biol. 278:457-479 for a listing ofhuman framework regions, which is incorporated by reference herein inits entirety). In a specific embodiment, an antibody of the inventioncomprises the framework region of the 26292, 32703, 32701, 32716antibody.

In a specific embodiment, the present invention provides for antibodiesthat bind to the IL3Rα chain, said antibodies comprising the amino acidsequence of the CDRs of the 26292, 32703, 32701, or 32716 antibodies andhuman murine framework regions. In another embodiment, the presentinvention provides for antibodies that bind to the IL3Rα chain, saidantibodies comprising the amino acid sequence of the CDRs of the 26292,32703, 32701 or 32716 antibodies and human or murine framework regionswith one or more amino acid substitutions at one, two, three, or more ofthe following residues: (a) rare framework residues that differ betweenthe murine antibody framework (i.e., donor antibody framework) and thehuman antibody framework (i.e., acceptor antibody framework); (b) Venierzone residues when differing between donor antibody framework andacceptor antibody framework; (c) interchain packing residues at theV_(H)/V_(L) interface that differ between the donor antibody frameworkand the acceptor antibody framework; (d) canonical residues that differbetween the donor antibody framework and the acceptor antibody frameworksequences, particularly the framework regions crucial for the definitionof the canonical class of the murine antibody CDR loops; (e) residuesthat are adjacent to a CDR; (g) residues capable of interacting with theantigen; (h) residues capable of interacting with the CDR; and (i)contact residues between the V_(H) domain and the V_(L) domain.

The present invention encompasses antibodies that bind to the IL3Rαchain, said antibodies comprising the amino acid sequence of the V_(H)domain and/or V_(L) domain of the 26292 antibody with mutations (e.g.,one or more amino acid substitutions) in the framework regions. Theframework regions of the V_(H) domain of the 26292 antibody are shown inFIG. 1B as the regions not underlined. The framework regions of theV_(L) domain of the 26292 antibody are shown in FIG. 1D as the regionsnot underlined. In an embodiment, an antibody that binds to the IL3Rαchain comprises the amino acid sequence of the V_(H) domain and/or V_(L)domain of the 32703 antibody with mutations (e.g., one or more aminoacid substitutions) in the framework regions. The framework regions ofthe V_(H) domain of the 32703 antibody are shown in FIG. 2B as theregions not underlined. The framework regions of the V_(L) domain of the32703 antibody are shown in FIG. 2D as the regions not underlined. Inanother embodiment, an antibody that binds to the IL3Rα chain comprisesthe amino acid sequence of the V_(H) domain and/or V_(L) domain of the32701 antibody with mutations (e.g., one or more amino acidsubstitutions) in the framework regions. The framework regions of theV_(H) domain of the 32701 antibody are shown in FIG. 3B as the regionsnot underlined. The framework regions of the V_(L) domain of the 32701antibody are shown in FIG. 3D as the regions not underlined. In anotherembodiment, an antibody that binds to the IL3Rα chain comprises theamino acid sequence of the V_(H) domain and/or V_(L) domain of the 32716antibody with mutations (e.g., one or more amino acid substitutions) inthe framework regions. The framework regions of the V_(H) domain of the32716 antibody are shown in FIG. 4B as the regions not underlined. Theframework regions of the V_(L) domain of the 32716 antibody are shown inFIG. 42D as the regions not underlined. In a specific embodiment, theamino acid substitutions in the framework region improve binding of theantibody to the IL3Rα chain.

In one embodiment, that binds to the IL3Rα chain, an antibody comprisesthe amino acid sequence of the V_(H) and/or V_(L) domains of the 26292antibody with mutations (e.g., one or more amino acid residuesubstitutions) in the hypervariable and framework regions. In anotherembodiment, an antibody that binds to the IL3Rα chain comprises theamino acid sequence of the V_(H) and/or V_(L) domains of the 32703antibody with mutations in the hypervariable and framework regions. Inanother embodiment, an antibody that binds to the IL3Rα chain comprisesthe amino acid sequence of the V_(H) and/or V_(L) domains of the 32701antibody with mutations in the hypervariable and framework regions. Inyet another embodiment, an antibody that binds to the IL3Rα chaincomprises the amino acid sequence of the V_(H) and/or V_(L) domains ofthe 32716 antibody with mutations in the hypervariable and frameworkregions. In a specific embodiment, the amino acid substitutions in thehypervariable and framework regions improve binding of the antibody tothe IL3Rα chain.

The present invention also provides antibodies of the invention thatbind to the IL3Rα chain that comprise constant regions known to those ofskill in the art. In one embodiment, the constant regions of an antibodyof the invention are human. In another embodiment, the constant regionsare derived from murine.

The present invention provides for antibodies that bind to the IL3Rαchain, the antibodies having an extended half-life in vivo and the useof such antibodies to produce an antibody conjugate of the invention. Ina specific embodiment, the present invention provides antibodies thatbind to the IL3Rα chain, which have a half-life in a subject, preferablya mammal and most preferably a human, of greater than about 2 minutes, 4minutes, 5 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 25minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 12 hours, 14 hours, 24 hours, 48hours, 1 week, 2 weeks, 1 month, 3 months, 6 months, 1 year 3 days,greater than 7 days, greater than 10 days, preferably greater than 15days, greater than 25 days, greater than 30 days, greater than 35 days,greater than 40 days, greater than 45 days, greater than 2 months,greater than 3 months, greater than 4 months, or greater than 5 months.In another embodiment, the present invention provides antibodies thatbind to the IL3Rα chain, which have a half-life in a subject, preferablya mammal and most preferably a human, of 2 minutes, 5 minutes, 10minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 12 hours, 14 hours, 24 hours, 48hours, 1 week, 2 weeks, 1 month, 3 months, 6 months, 1 year 3 days,greater than 7 days, greater than 10 days, preferably greater than 15days, greater than 25 days, greater than 30 days, greater than 35 days,greater than 40 days, greater than 45 days, greater than 2 months,greater than 3 months, greater than 4 months, or greater than 5 months.

To prolong the serum circulation of antibody conjugates (e.g.,monoclonal antibodies, single chain antibodies, Fv fragments, and Fabfragments) in vivo, for example, inert polymer molecules such as highmolecular weight polyethylene glycol (PEG) can be attached to theantibodies with or without a multifunctional linker either throughsite-specific conjugation of the PEG to the amino or carboxyl terminusof the antibodies (whichever end is not conjugated to the cytotoxicagent) or via epsilon-amino groups present on lysine residues. Linear orbranched polymer derivatization that results in minimal loss ofbiological activity will be used. The degree of conjugation can beclosely monitored by SDS-PAGE and mass spectrometry to ensure properconjugation of PEG molecules to the antibodies. Unreacted PEG can beseparated from antibody-PEG conjugates by size-exclusion or ion-exchangechromatography. Polyethylene glycol-derivatized antibodies can be testedfor binding activity as well as for in vivo efficacy using methodswell-known to those of skill in the art, for example, by immunoassaysdescribed herein.

Antibodies having an increased half-life in vivo can also be generatedby introducing one or more amino acid modifications (i.e.,substitutions, insertions, or deletions) into an IgG constant domain, orFcRn binding fragment thereof (preferably a Fc or hinge Fc domainfragment). See, e.g., International Publication No. WO 98/23289;International Publication No. WO 97/34631; International Publication No.WO 02/060919; and U.S. Pat. No. 6,277,375, each of which is incorporatedherein by reference in its entirety.

Furthermore, antibodies can be conjugated to albumin in order to makethe antibody more stable in vivo or have a longer half-life in vivo. Thetechniques are well-known in the art; see, e.g., InternationalPublication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and EuropeanPatent No. EP 413,622, all of which are incorporated herein byreference.

The present invention provides for panels of antibodies that bind to theIL3Rα chain. In specific embodiments, the invention provides for panelsof antibodies having different affinities for the IL3Rα chain, differentspecificities for the IL3Rα chain, and/or different dissociation rates.The invention provides panels of at least 10, preferably at least 25, atleast 50, at least 75, at least 100, at least 125, at least 150, atleast 175, at least 200, at least 250, at least 300, at least 350, atleast 400, at least 450, at least 500, at least 550, at least 600, atleast 650, at least 700, at least 750, at least 800, at least 850, atleast 900, at least 950, or at least 1000 antibodies. Panels ofantibodies can be used, for example, in 96 well plates for assays suchas ELISAs and cytotoxicity assays.

5.3 Cytotoxic Agents

Any cytotoxic agent or otherwise anticellular agent known to one ofskill in the art can be used to produce the antibody conjugates of theinvention. A cytotoxic agent includes any agent that is detrimental tocells. Exemplary cytotoxic agents include chemotherapeutic agents,radioisotopes, cytotoxins such as cytostatic or cytocidal agents, orother anticellular agents, including known therapeutic agents.

Non-limiting examples of cytotoxic agents include antimetabolites (e.g.,cytosine arabinoside, aminopterin, methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, and 5-fluorouracil decarbazine); alkylatingagents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan,carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C,cis-dichlorodiammine-platinum (II) (CDDP), and cisplatin); vincaalkaloid; anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin); antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)); calicheamicin; CC-1065and derivatives thereof; auristatin molecules (e.g., auristatin PHE,bryostatin-1, and dolastatin-10; see Woyke et al., Antimicrob. AgentsChemother. 46:3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother.45:3580-4 (2001), Mohammad et al., Anticancer Drugs 12:735-40 (2001),Wall et al., Biochem. Biophys. Res. Commun. 266:76-80 (1999), Mohammadet al., Int. J. Oncol. 15:367-72 (1999), all of which are incorporatedherein by reference); DNA-repair enzyme inhibitors (e.g., etoposide ortopotecan); kinase inhibitors (e.g., compound ST1571, imatinib mesylate(Kantarjian et al., Clin Cancer Res. 8(7):2167-76 (2002)); demecolcine;and other cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,dihydroxy anthracenedione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologues thereof and thosecompounds disclosed in U.S. Pat. Nos. 6,245,759, 6,399,633, 6,383,790,6,335,156, 6,271,242, 6,242,196, 6,218,410, 6,218,372, 6,057,300,6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995,5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, 5,587,459);farnesyl transferase inhibitors (e.g., R115777, BMS-214662, and thosedisclosed by, for example, U.S. Pat. Nos. 6,458,935, 6,451,812,6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541,6,410,539, 6,403,581, 6,399,615, 6,387,905, 6,372,747, 6,369,034,6,362,188, 6,342,765, 6,342,487, 6,300,501, 6,268,363, 6,265,422,6,248,756, 6,239,140, 6,232,338, 6,228,865, 6,228,856, 6,225,322,6,218,406, 6,211,193, 6,187,786, 6,169,096, 6,159,984, 6,143,766,6,133,303, 6,127,366, 6,124,465, 6,124,295, 6,103,723, 6,093,737,6,090,948, 6,080,870, 6,077,853, 6,071,935, 6,066,738, 6,063,930,6,054,466, 6,051,582, 6,051,574, and 6,040,305); topoisomeraseinhibitors (e.g., camptothecin, irinotecan, SN-38, topotecan,9-aminocamptothecin, GG211 (GI147211), DX-8951f, IST-622, rubitecan,pyrazoloacridine, XR5000, saintopin, UCE6, UCE1022, TAN-1518A, TAN1518B, KT6006, KT6528, ED-110, NB-506, ED-110, NB-506, andrebeccamycin); bulgarein; DNA minor groove binders such as Hoechst dye33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine;coralyne; beta-lapachone; BC-4-1; antisense oligonucleotides (e.g.,those disclosed in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834,5,734,033, and 5,618,709); adenosine deaminase inhibitors (e.g.,fludarabine phosphate and 2-chlorodeoxyadenosine); and pharmaceuticallyacceptable salts, solvates, clathrates, and prodrugs thereof.

Other examples of cytotoxic agents which can be used to produce theantibody conjugates of the invention include antimitotic drugs, such asauristatin, derivatives of auristatin, monomethylauristatin, andderivatives of monomethylauristatin, such as monomethylauristatin F andmonomethylauristatin E.

Other examples of cytotoxic agents which can be used to produce theantibody conjugates of the invention include maytansine (Cassady et al.,2004, Chem. Pharm. Bull. 52(1):1-26), and maytansine derivatives such asDM1 (Tassone et al., 2008, Blood 104(12): 3688-3696; Erickson et al.,2006, Cancer Res 66(8) 4426-4433) and DM4 (Erickson et al., 2006, CancerRes 66(8) 4426-4433)

Other examples of cytotoxic agents which can be used to produce theantibody conjugates of the invention are single-walled carbon nanotubes(Gannon et al., 2007 Cancer, 110:2654-2665).

In one embodiment, the IL3Rα antibody is conjugated to a radioactivemetal ion, such as the alpha-emitters ²¹¹astatine, ²¹²bismuth,²¹³bismuth; the beta-emitters ¹³¹iodine, ⁹⁰yttrium, ¹⁷⁷lutetium,¹⁵³samarium, and ¹⁰⁹palladium; or macrocyclic chelators useful forconjugating radiometal ions, including but not limited to, ¹³¹indium,¹³¹L, ¹³¹yttrium, ¹³¹holmium, ¹³¹samarium, to polypeptides or any ofthose listed supra. In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA),which can be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug.Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol.26(8):943-50, each incorporated by reference in their entireties.

In a specific embodiment, the IL3Rα antibody is conjugated to aproteinaceous agent that modifies a given biological response and leadsto cytotoxicity. In one embodiment, the IL3Rα antibody is conjugated toa plant-, fungus-, or bacteria-derived toxin. Non-limiting examples ofsuch toxins include A chain toxins, ribosome inactivating proteins,ricin A, deglycosylated ricin A chain, abrin, alpha sarcin, aspergillin,restrictocin, ribonucleases, diphtheria toxin, bacterial endotoxin,saporin toxin, Granzyme B or the lipid A moiety of bacterial endotoxin,cholera toxin, or Pseudomonas exotoxin and derivatives and variantsthereof.

Fragments, analogs, and derivatives of proteinaceous cytotoxins can beuseful in the present invention provided that when fused to the IL3Rαantibody portion of the conjugate, such fragments, analogs, andderivatives allow the conjugate to bind native IL3Rαexpressed on thesurface of a cell. Preferably, the binding kinetics of the fragments,analogs, or derivatives remain the same or vary only by not more than25%. The cytotoxin may be from any species. The nucleotide and/or aminoacid sequences of cytotoxins can be found in the literature or publicdatabases, or the nucleotide and/or amino acid sequences can bedetermined using cloning and sequencing techniques known to one of skillin the art. In some embodiments, the cytotoxin is derived from mammals.In other embodiments, the cytotoxin is derived from bacteria. In yetother embodiments, the cytotoxin is derived from fungi. In a preferredembodiment, the cytotoxin is Pseudomonas exotoxin A (PE), or an analog,derivative, or a fragment thereof.

In one embodiment of the invention, the proteinaceous cytotoxincomprises an amino acid sequence which contains at least oneconservative amino acid substitution, but not more than 40 conservativeamino acid substitutions, even more preferably, not more than 30conservative amino acid substitutions, still more preferably, not morethan 20 conservative amino acid substitutions, and still even morepreferably, not more than 10 conservative amino acid substitutionsrelative to the native amino acid sequence of the chosen fragment (e.g.,the native PE or diphtheria toxin amino acid sequence), which result ina silent change, i.e., no change in an activity necessary forcytotoxicity in the context of the conjugate. In another embodiment ofthe invention, a cytotoxin comprises an amino acid sequence thatcontains at least one conservative amino acid substitution in the chosenfragment; but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservativeamino acid substitutions relative to the native amino acid sequence(e.g., the native PE or diphtheria toxin amino acid sequence), whichresult in a silent change. In yet another embodiment, a cytotoxicpolypeptide comprises an amino acid sequence that contains one or moreconservative substitutions or a combination of non-conservative andconservative amino acid substitutions relative to the native amino acidsequence of the chosen fragment, which results in a silent change.

To improve or alter the characteristics of cytotoxic polypeptides,protein engineering may be employed. Recombinant DNA technology known tothose skilled in the art can be used to create novel mutant proteins or“muteins” including single or multiple amino acid substitutions,deletions, additions, or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions. For instance, for many proteins, it is known inthe art that one or more amino acids may be deleted from the aminoterminus or carboxyl terminus without substantial loss of biologicalfunction. Exemplary cytotoxin variants suitable for forming theconjugates of the invention are found hereinbelow.

In a specific embodiment, a cytotoxic polypeptide is at least 50%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% identical to the amino acid sequence ofchosen fragment of the native cytotoxin (e.g., the native PE ordiphtheria toxin amino acid sequence).

5.3.1 Pseudomonas Exotoxin and Variants Thereof

Pseudomonas exotoxin A (PE) is an extremely active 66 kDa protein (613or 615 amino acids) secreted by Pseudomonas aeruginosa. The amino acidsequence of PE can be found in the GenBank database (see, e.g.,Accession No. 1IKQA). It is composed of three discrete structuraldomains: an amino-terminal receptor binding domain (domain Ia; aminoacids 1-252), a middle translocation domain (domain II; amino acids253-364), a minor domain (domain Ib; amino acids 365-399 or 365-404),and a carboxyl-terminal ADP-ribosylating domain (domain III; amino acids400-613/615 or 405-613/615). Upon binding its receptor on the surface ofa eukaryotic cell, it is internalized by endocytosis. The toxin is thenactivated by proteolytic cleavage between amino acids 279 and 280, andforms a channel through which the enzymatic domain translocates to thecytosol. Once in the cytosol, PE inhibits protein synthesis, and thusexerts its cytotoxic effect, by catalyzing the irreversibleADP-ribosylation (transfer of the ADP ribosyl moiety from oxidized NAD)of translational elongation factor 2 (EF-2). See, e.g., U.S. Pat. No.4,892,827, and Seetharam et al., “Increased Cytotoxic Activity ofPseudomonas Exotoxin and Two Chimeric Toxins Ending in KDEL,” J. Biol.Chem. 266:17376-17381 (1991), which are incorporated by reference hereinin their entirety.

Pseudomonas exotoxin-containing immunotoxins were previously constructedby first reacting native PE with iminothiolane. This reaction servedboth to introduce two new sulfhydryl groups used for coupling the anantibody to the toxin, and to inactivate the binding of PE to its ownreceptor. This approach relied on the chemical inactivation ofPE-binding sites in order to minimize undesirable side effects of theimmunotoxin binding to cells with PE receptors. Although this approachhas been successful in killing specific cells in tissue culture andtargeting tumors in mice, its therapeutic utility is limited as itcannot be administered in high doses. Therefore, catalytically-activefragments of PE are designed to overcome this limitation, facilitatingthe development of immunotoxins with high potency and low toxicity. Forexample, PE40 (Pseudomonas exotoxin fragment of 40 kDa is a fragment ofPE that comprises the entire protein except for the receptor-bindingdomain (domain Ia); in immunotoxins, domain Ia can be either deleted orsubstituted. Moreover, additional fragments of PE have been developed,to yield cytotoxins with greater potency. These include PE37, which is afragment that lacks domain Ia and is mutated such that it does not needto undergo proteolytic cleavage for activation (e.g., is deleted foramino acids 1-279; other mutations that result in a constitutivelyactive fragment have been contemplated. PE35 additionally lacks part ofdomain Ib; other mutations in which domain Ib, in its entirety or atleast the amino-terminal half of it, is deleted or substituted have beendeveloped. PE38 is a fragment is deleted for domain Ia and amino acids365-380 of Ib. Any combination of the modifications of PE describedabove, or other modifications that allow it to retain its cytotoxicactivity and specificity when conjugated to an IL3Rα antibody, arecontemplated. Other variants of PE that can be used in the presentinvention include those in which the carboxyl terminus is mutated fromArg-Glu-Asp-Leu-Lys (REDLK) to KDEL or KDEL repeats (e.g., (KDEL)2 or(KDEL)3). The KDEL mutation has been shown to increase the activity ofimmunotoxins by increasing their binding to the intracellular KDELreceptor and thereby increasing the amount of immunotoxin that reachesthe cytosol. All of the above fragments of PE can be mutated so thatthey have the KDEL sequence at there carboxyl terminus. See, e.g.,Seetharam et al., “Increased Cytotoxic Activity of Pseudomonas Exotoxinand Two Chimeric Toxins Ending in KDEL,” J. Biol. Chem. 266:17376-17381(1991) and U.S. Pat. Nos. 4,892,827, 5,696,237, 5,747,654, 5,863,745,5,980,895, 6,051,405, 6,074,644, 6,147,203, and 6,558,672, which areincorporated by reference herein in their entirety.

Fragments, analogs, and derivatives of PE can be useful in the presentapplication. In some embodiments, the cytotoxic moiety of the inventionconsists of full-length PE, i.e., the receptor binding, translocation,minor, and catalytic domains. In other embodiments, the cytotoxic moietyconsists of the fragments PE35, PE37, PE38, or PE40. In preferredembodiments, the cytotoxic moiety of the invention consists of PE38.

Moreover, the antibody moiety of PE-containing immunotoxins can besubstituted into different parts of PE, so that the above PE variants(including KDEL-containing variants) contain an IL3Rα antibody orfragment thereof (e.g., but not by limitation, scFv, dsFv wherein V_(H)is in the same polypeptide as PE, or dsFv wherein V_(L) is in the samepolypeptide as PE) in place of domain Ia, in place of amino acids 1-279,or in place of all or part of domain Ib, as exemplary embodiments of theinvention. In other embodiments of the invention, the antibody isinserted at the carboxyl terminus of the PE variant, wherein the nativePE carboxyl-terminal amino acids 604/607-613/615 are moved to orrepeated at the end of the molecule, so that the native carboxylterminus of PE is retained (necessary for its cytotoxic activityalthough unrelated to its ADP ribosylation activity). In anotherembodiment, the antibody is inserted at the carboxyl terminus of the PEvariant, wherein the native PE carboxyl-terminal amino acids604/607-613/615 are moved to or repeated at the end of the molecule andthe REDLK terminus is replaced with KDEL. An antibody or fragmentthereof comprising two polypeptide chains, one of which is in the samepolypeptide as the PE variant, can also be inserted at the carboxylterminus of PE as long as one of the chains contains PE amino acids604/607-613/615, or the REDLK-to-KDEL substitution, at its carboxylterminus. See, e.g., Seetharam et al., “Increased Cytotoxic Activity ofPseudomonas Exotoxin and Two Chimeric Toxins Ending in KDEL,” J. Biol.Chem. 266:17376-17381 (1991) and U.S. Pat. Nos. 4,892,827, 5,696,237,5,747,654, 5,863,745, 5,980,895, 6,051,405, 6,074,644, 6,147,203, and6,558,672, which are incorporated by reference herein in their entirety.

Other fragments, analogs, and derivatives of PE can be useful in thepresent invention provided that when fused to the anti-IL3Rα antibodyportion of the conjugate, such fragments, analogs, and derivatives donot prevent the IL3Rα antibody from binding to IL3Rαexpressed on thesurface of a cell. Preferably, the binding kinetics of the fragments,analogs, or derivatives remain the same or vary only by not more than25%. The PE polypeptide may be obtained from any species, heterologouslyor natively expressed. The nucleotide and/or amino acid sequences of PEpolypeptides can be found in the literature or public databases, or thenucleotide and/or amino acid sequences can be determined using cloningand sequencing techniques known to one of skill in the art. In someembodiments, PE is expressed in Escherichia coli.

In one embodiment of the invention, a PE polypeptide comprises an aminoacid sequence that contains at least one conservative amino acidsubstitution, but not more than 50 conservative amino acidsubstitutions, even more preferably, not more than 40 conservative aminoacid substitutions, still more preferably, not more than 30 conservativeamino acid substitutions, and still even more preferably, not more than20 conservative amino acid substitutions relative to the native aminoacid sequence of the chosen fragment (e.g., the native amino acidsequence of PE38), which result in a silent change, i.e., no change inactivity. In another embodiment of the invention, a PE polypeptidecomprises an amino acid sequence that contains at least one conservativeamino acid substitution; but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or1 conservative amino acid substitutions relative to the native aminoacid sequence of the chosen fragment (e.g., the native PE38 amino acidsequence), which result in a silent change. In yet another embodiment, aPE polypeptide comprises an amino acid sequence that contains one ormore conservative substitutions or a combination of non-conservative andconservative amino acid substitutions relative to the native amino acidsequence of the chosen fragment, which results in a silent change.

To improve or alter the characteristics of PE polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins” including single or multiple amino acid substitutions,deletions, additions, or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

In another embodiment, a PE polypeptide is at least 50%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% identical to the native amino acid sequence of a chosenfragment (e.g., the native sequence of PE38).

5.4 Therapeutic Agents

Any therapeutic agent known to one of skill in the art can be used toproduce the antibody conjugates of the invention. A therapeutic agentincludes any agent that, when conjugated to an antibody or fragmentthereof of the invention, can be used to treat cancer. In certainembodiments, a cytotoxic agent as exemplified in section 5.3 can be atherapeutic agent.

An antibody of the invention can be conjugated to therapeutic agentssuch as macrocyclic chelators useful for conjugating radiometal ions. Incertain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo, etal., 1998, Clin Cancer Res 4:2483-90; Peterson, et al., 1999, BioconjugChem 10:553; and Zimmerman, et al., 1999, Nucl Med Biol 26:943-50 eachincorporated by reference in their entireties.

Examples of useful therapeutic radioisotopes (ordered by atomic number)include 47Sc, 67Cu, 90Y, 109Pd, 125I, 131I, 186Re, 188Re, 199 Au, 211At,212Pb and 217Bi. These atoms can be conjugated to the peptide directly,indirectly as part of a chelate, or, in the case of iodine, indirectlyas part of an iodinated Bolton-Hunter group. The radioiodine can beintroduced either before or after this group is coupled to the peptidecompound.

Examples of therapeutic agents which can be used to produce the antibodyconjugates of the invention include, Bcl-2 family inhibitors and Bcl-2inhibitors, including ABT-737.

Other examples of therapeutic agents which can be used to produce theantibody conjugates of the invention include nanoparticles, such asperfluorocarbon nanoparticles, (Tran et al., 2007, Int. J. Nanomedicine2(4):515-526) and paramagnetic nanoparticles (Cyrus et al., 2008,Arterioscler Thromb Vasc Biol (on-line publication)).

An antibody or fragment thereof may be conjugated to a therapeutic agentthat modifies a given biological response. Therapeutic agents are not tobe construed as limited to classical chemical therapeutic agents. Forexample, the therapeutic agent may be a protein or polypeptidepossessing a desired biological activity. Such proteins may include, forexample, a toxin such as abrin, ricin A, pseudomonas exotoxin, choleratoxin, or diphtheria toxin; a protein such as tumor necrosis factor,α-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, e.g., TNF-α, AIM I(see, International Publication No. WO 97/33899 herein incorporated byreference in its entirety), AIM II (see, International Publication No.WO 97/34911 herein incorporated by reference in its entirety), FasLigand (Takahashi, et al., 1994, J Immunol, 6:1567 herein incorporatedby reference in its entirety), and VEGI (see, International PublicationNo. WO 99/23105 herein incorporated by reference in its entirety), athrombotic agent or an anti-angiogenic agent, e.g., angiostatin orendostatin; or, a biological response modifier such as, for example, alymphokine (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)), or agrowth factor (e.g., growth hormone (“GH”)).

Antibodies of the invention or fragments thereof can also be conjugatedto Lectins. Lectins are proteins, commonly derived from plants, thatbind to carbohydrates. Among other activities, some lectins are toxic.Some of the most cytotoxic substances known are protein toxins ofbacterial and plant origin (Frankel et al., Ann Rev Med 37:125-142(1986) herein incorporated by reference in its entirety). Thesemolecules binding the cell surface and inhibit cellular proteinsynthesis. The most commonly used plant toxins are ricin and abrin; themost commonly used bacterial toxins are diphtheria toxin and Pseudomonasexotoxin A. In ricin and abrin, the binding and toxic functions arecontained in two separate protein subunits, the A and B chains. Thericin B chain binds to the cell surface carbohydrates and promotes theuptake of the A chain into the cell. Once inside the cell, the ricin Achain inhibits protein synthesis by inactivating the 60S subunit of theeukaryotic ribosome Endo, et al., J Biol Chem 262: 5908-5912 (1987)herein incorporated by reference in its entirety). Other plant derivedtoxins, which are single chain ribosomal inhibitory proteins, includepokeweed antiviral protein, wheat germ protein, gelonin, dianthins,momorcharins, trichosanthin, and many others (Strip, et al., FEBS Lett195:1-8 (1986) herein incorporated by reference in its entirety).Diphtheria toxin and Pseudomonas exotoxin A are also single chainproteins, and their binding and toxicity functions reside in separatedomains of the same protein Pseudomonas exotoxin A has the samecatalytic activity as diphtheria toxin. Ricin has been usedtherapeutically by binding its toxic a-chain, to targeting moleculessuch as Abs to enable site-specific delivery of the toxic effect.Bacterial toxins have also been used as anti-tumor conjugates. Asintended herein, a toxic peptide chain or domain is conjugated to acompound of this invention and delivered in a site-specific manner to atarget site where the toxic activity is desired, such as a metastaticfocus.

5.5 Methods for Producing IL3RαAntibody Conjugates

Techniques for conjugating cytotoxic agents or otherwise anticellularmoieties to antibodies are well known, e.g., Amon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy,” inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery,” in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in MonoclonalAntibodies 84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabelled Antibody In Cancer Therapy,” inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58; each of which is incorporated herein byreference in its entirety.

Methods for fusing or conjugating proteins, polypeptides, or peptides toan antibody are known in the art, and can be used to conjugateproteinaceous cytotoxins to antibodies, or for connecting cytotoxins toantibodies through a peptide linker. See, e.g., U.S. Pat. Nos.5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946;European Patent Nos. EP 307,434 and EP 367,166; InternationalPublication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991,Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J.Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA89:11337-11341 (said references are incorporated herein by reference intheir entireties).

The conjugates of the present invention can be made by standardrecombinant DNA techniques or by protein synthetic techniques, e.g., byuse of a peptide synthesizer. For example, a nucleic acid moleculeencoding a conjugate of the invention can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primersthat give rise to complementary overhangs between two consecutive genefragments, which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, e.g., Current Protocols inMolecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).

The nucleotide sequences encoding a conjugate or portion thereof of theinvention (anti-IL3Rα antibody and/or proteinaceous cytotoxin sequences,such as but not limited to Pseudomonas exotoxin or diphtheria toxin) maybe obtained from any information available to those of skill in the art(i.e., from GenBank, the literature, or by routine cloning). Thenucleotide sequence coding for a conjugate, or for the antibody orcytotoxin moiety of the conjugate, can be inserted into an appropriateexpression vector, i.e., a vector that contains the necessary elementsfor the transcription and translation of the inserted protein-codingsequence. A variety of host-vector systems may be utilized in thepresent invention to express the protein-coding sequence. These includebut are not limited to mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); microorganisms such as yeast containing yeastvectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA,or cosmid DNA. The expression elements of vectors vary in theirstrengths and specificities. Depending on the host-vector systemutilized, any one of a number of suitable transcription and translationelements may be used.

The expression of a conjugate, or a portion thereof, of the inventionmay be controlled by any promoter or enhancer element known in the art.Promoters that may be used to control expression of a conjugate or itsparts include, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, 1981, Nature 290:304-310), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al.,1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner etal., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatorysequences of the metallothionein gene (Brinster et al., 1982, Nature296:39-42), the tetracycline (Tet) promoter (Gossen et al., 1995, Proc.Nat. Acad. Sci. USA 89:5547-5551); prokaryotic expression vectors suchas the β-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl.Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al.,1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25; see also “Useful proteinsfrom recombinant bacteria” in Scientific American, 1980, 242:74-94);plant expression vectors comprising the nopaline synthetase promoterregion (Herrera-Estrella et al., Nature 303:209-213) or the cauliflowermosaic virus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res.9:2871), and the promoter of the photosynthetic enzyme ribulosebiphosphate carboxylase (Herrera-Estrella et al., 1984, Nature310:115-120); promoter elements from yeast or other fungi such as theGAL4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter, andthe following animal transcriptional control regions, which exhibittissue specificity and have been utilized in transgenic animals:elastase I gene control region, which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology7:425-515); insulin gene control region, which is active in pancreaticbeta cells (Hanahan, 1985, Nature 315:115-122); immunoglobulin genecontrol region, which is active in lymphoid cells (Grosschedl et al.,1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); mouse mammarytumor virus control region, which is active in testicular, breast,lymphoid, and mast cells (Leder et al., 1986, Cell 45:485-495); albumingene control region, which is active in liver (Pinkert et al., 1987,Genes and Devel. 1:268-276); alpha-fetoprotein gene control region,which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsingene control region, which is active in the liver (Kelsey et al., 1987,Genes and Devel. 1:161-171); beta-globin gene control region, which isactive in myeloid cells (Mogram et al., 1985, Nature 315:338-340;Kollias et al., 1986, Cell 46:89-94); myelin basic protein gene controlregion, which is active in oligodendrocyte cells in the brain (Readheadet al., 1987, Cell 48:703-712); myosin light chain-2 gene controlregion, which is active in skeletal muscle (Sani, 1985, Nature314:283-286); neuronal-specific enolase (NSE), which is active inneuronal cells (Morelli et al., 1999, Gen. Virol. 80:571-83);brain-derived neurotrophic factor (BDNF) gene control region, which isactive in neuronal cells (Tabuchi et al., 1998, Biochem. Biophysic. Res.Com. 253:818-823); glial fibrillary acidic protein (GFAP) promoter,which is active in astrocytes (Gomes et al., 1999, Braz J Med Biol Res32(5):619-631; Morelli et al., 1999, Gen. Virol. 80:571-83); andgonadotropic releasing hormone gene control region, which is active inthe hypothalamus (Mason et al., 1986, Science 234:1372-1378). In aspecific embodiment, the expression of a conjugate of the invention isregulated by a constitutive promoter. In another embodiment, expressionis regulated by an inducible promoter. In another embodiment, expressionis regulated by a tissue-specific promoter.

In a specific embodiment, a vector is used that comprises a promoteroperably linked to a conjugate-encoding nucleic acid (or nucleic acidthat encodes a portion of the conjugate), one or more origins ofreplication, and, optionally, one or more selectable markers (e.g., anantibiotic resistance gene).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, polypeptide(s) of the conjugate may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted intothe adenovirus genome by in vitro or in vivo recombination. Insertioninto a non-essential region of the viral genome (e.g., region E1 or E3)will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule and/or proteinaceous cytotoxic moietyin infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci.USA 81:355-359). Specific initiation signals may also be required forefficient translation of inserted fusion protein coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBittner et al., 1987, Methods in Enzymol. 153:51-544).

Expression vectors containing inserts of a gene encoding a conjugate ora portion thereof can be identified by three general approaches: (a)nucleic acid hybridization; (b) presence or absence of “marker” genefunctions; and (c) expression of inserted sequences. In the firstapproach, the presence of a gene encoding a conjugate or a portionthereof in an expression vector can be detected by nucleic acidhybridization using probes comprising sequences that are homologous tothe inserted gene. In the second approach, the recombinant vector/hostsystem can be identified and selected based upon the presence or absenceof certain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of a nucleotidesequence encoding a conjugate or a portion thereof into the vector. Forexample, if the nucleotide sequence encoding a segment(s) of a conjugateis inserted within the marker gene sequence of the vector, recombinantscontaining the gene encoding the conjugate insert can be identified bythe absence of the marker gene function. In the third approach,recombinant expression vectors can be identified by assaying the geneproduct (e.g., conjugate) expressed by the recombinant. Such assays canbe based, for example, on the physical or functional properties of theconjugate in in vitro assay systems, e.g., binding to IL3Rα, killingIL3Rα-expressing cells, competing with other IL3Rα antibodies or withIL-3 for binding to IL3Rα, etc.

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered conjugates or componentsthereof may be modulated. Furthermore, different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,phosphorylation) of proteins. Appropriate cell lines or host systems canbe chosen to ensure the desired modification and processing of theforeign protein expressed. For example, expression in yeast will producea glycosylated product and expression in a bacterial system will producean unglycosylated product. Eukaryotic host cells that possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include, but are not limited to, CHO, VERY, BHK,HeLa, COS, MDCK, 293, 3T3, WI38, NS0. Furthermore, different vector/hostexpression systems may affect processing reactions to different extents.

For long-term, high-yield production of recombinant conjugates, stableexpression is preferred. For example, cell lines that stably express theconjugate or a portion thereof of the invention may be engineered.Rather than using expression vectors that contain viral origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedium, and then are switched to a selective medium. The selectablemarker in the recombinant plasmid confers resistance to the selectionand allows cells to stably integrate the plasmid into their chromosomesand grow to form foci, which in turn can be cloned and expanded intocell lines. This method may advantageously be used to engineer celllines that express a conjugate of the invention. Such engineered celllines may be particularly useful in screening and evaluating compoundsthat affect the activity of an immunotoxin conjugate binding to IL3Rα orcytotoxin action.

A number of selection systems may be used, including but not limited to:the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30:147) genes.

Once a conjugate or a portion thereof (i.e., the antibody, a chain ofthe antibody fused to a cytotoxin, the cytotoxin, etc.) of the inventionhas been produced by recombinant expression or by chemical synthesis, itmay be purified by methods known in the art for purification and/orrefolding of proteins, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antibodyfor Protein A, or the antibody for IL3Rα, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Expression inbacteria may result in the antibody-cytotoxin conjugate being secretedinto the periplasm. In such a case, proteins are purified using standardchromatographic techniques, for example, a two-step procedure thatinvolves a Mono Q (Pharmacia LKB Biotechnology Inc.) anion exchangecolumn and TSK-250 gel filtration column chromatography.

Often, expression in bacteria causes functional proteins, such as theIL3Rα antibody conjugates of the invention, to be localized in inclusionbodies. This requires that the proteins are solubilized using strongdenaturants and subsequently refolded. In the solubilization step, areducing agent must be present to dissolve disulfide bonds as is wellknown in the art. An exemplary buffer with a reducing agent is: 0.1 MTris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Re-oxidation of protein disulfide bonds can be effectively catalyzed inthe present of low molecular weight thiol reagents in reduced andoxidized form. Renaturation is typically accomplished by rapid dilution(e.g., 100 fold) of the denatured and reduced protein into refoldingbuffer. An exemplary buffer is 0.1 M Tris pH 8.0, 0.5 M L-arginine, 8 mMoxidized glutathione, and 2 mM EDTA. Another buffer isphosphate-buffered saline. Other techniques known to those skilled inthe art may be used. When the antibody moiety of the immunotoxincontains multiple chains (e.g., dsFv fragments consisting of modifiedV_(H) and V_(L), one of which is continuous with a proteinaceouscytotoxin such as PE), each chain can be solubilized separately and thencombined in the refolding solution. A preferred yield is obtained whenthese two proteins are mixed in a molar ratio such that a molar excessof one protein over the other does not exceed a 5 fold excess. It isdesirable to add excess oxidized glutathione or other oxidizing lowmolecular weight compounds to the refolding solution after theredox-shuffling is completed. Alternatively, the final oxidation can beomitted and the refolding carried out at pH 9.5. Renatured material canthen be purified using standard chromatography techniques, for example,a three-step column chromatographic procedure involving Q-Sepharose,Mono Q, and TSK 250 gel filtration can be used to obtain highly purifiedmonomeric fusion proteins. See, e.g., Seetharam et al., “IncreasedCytotoxic Activity of Pseudomonas Exotoxin and Two Chimeric ToxinsEnding in KDEL,” J. Biol. Chem. 266:17376-17381 (1991) and U.S. Pat. No.6,074,644, which are incorporated by reference herein in their entirety.

In a specific embodiment of the invention, a scFv-PE38 immunotoxin isconstructed and purified. Total cellular RNA is isolated from hybridomacells expressing the desired monoclonal antibodies using methods knownto those skilled in the art, for example, using the Qiagen RNeasy minikit. V_(H) and V_(L) cDNAs of the monoclonal antibodies can be obtainedby a RACE method using SMART RACE cDNA amplification kit (Clontech) asdescribed in Pastan I, Beers R, Bera T K. Recombinant immunotoxins inthe treatment of cancer. Methods Mol Biol 2004; 248:503-18, or othermethod for preparation of cDNA known to those of skill in the art.Prepared cDNAs are then used as the template for PCR reactions toamplify the desired V_(H) and V_(L) fragments. The PCR products can thenbe cloned into an appropriate vector, either directly into a vectorcontaining the cytotoxin, or transferred to a shuttle vector, such aspCR4®-TOPO®, using the TOPO TA cloning kit (Invitrogen). Clones for eachchain should be sequenced to exclude the possibility of PCR error. Theobtained sequences should be confirmed again by comparing sequences in,e.g., a public database such as GenBank. The V_(H) and V_(L) chains canthen be assembled into a single chain Fv (scFv) and fused to PE38 asdescribed in Pastan I, Beers R, Bera TK. Recombinant immunotoxins in thetreatment of cancer. Expression and purification of scFv-PE38 is asdescribed in Pastan I, Beers R, Bera T K. Recombinant immunotoxins inthe treatment of cancer. Methods Mol Biol 2004; 248:503-18, which isincorporated herein by reference in its entirety.

In another embodiment, the PE cytotoxin can be modified so that itscarboxyl terminus contains the KDEL sequence. For example, a KDELvariant can be constructed by replacing the PE38 portion from theantibody-cytotoxin conjugate expression plasmid with PE38 containingKDEL, as described in Kreitman R J, Margulies I, Stetler-Stevenson M, etal. Cytotoxic activity of disulfide-stabilized recombinant immunotoxinRFB4(dsFv)-PE38 (BL22) toward fresh malignant cells from patients withB-cell leukemias. Clin Cancer Res 2000; 6:1476-87, which is incorporatedherein by reference in its entirety.

5.6 Methods for Producing Antibodies

Antibodies that bind to an antigen can be produced by any method knownin the art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques. In someembodiments, an immunogen that comprises an epitope unique to a proteincomponent is used to generate an antibody specific for the component.

Polyclonal antibodies specific for an antigen can be produced by variousprocedures well-known in the art. As a non-limiting example, the antigen(i.e., human IL3Rα) can be administered to various host animalsincluding, but not limited to, rabbits, mice, rats, etc. to induce theproduction of sera containing polyclonal antibodies specific for thehuman antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to Freund's (complete and incomplete), mineral gels suchas aluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Köhler & Milstein, 1975,Nature 256:495-497; Pasqualini & Arap, 2004, PNAS USA 101:257-259;Steinitz et al., 1977, Nature 269:420-422; Vollmers et al., 1989, CancerRes. 49:2471-2476; Vollmers & Brandlein, 2002 Hum. Antibodies11(4):131-142; Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in:Monoclonal Antibodies and T Cell Hybridomas 563 681 (Elsevier, N.Y.,1981) (said references incorporated by reference in their entireties).The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. Briefly,mice can be immunized with a non-murine antigen and once an immuneresponse is detected, e.g., antibodies specific for the antigen aredetected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of binding IL3Rα.Ascites fluid, which generally contains high levels of antibodies, canbe generated by immunizing mice with positive hybridoma clones.

The present invention provides methods of generating monoclonalantibodies as well as antibodies produced by the method comprisingculturing a hybridoma cell (e.g., the hybridoma that expresses 26292,32703, 32701 or 32716 monoclonal antibody) secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with a non-murine antigenwith myeloma cells and then screening the hybridomas resulting from thefusion for hybridoma clones that secrete an antibody able to bind to theantigen.

Antibody fragments that recognize specific particular epitopes on IL3Rαmay be generated by any technique known to those of skill in the art.For example, total cellular RNA is isolated from hybridoma cellsexpressing the desired monoclonal antibodies using methods known tothose skilled in the art, e.g., using the Qiagen RNeasy mini kit. V_(H)and V_(L) cDNAs of the monoclonal antibodies can be obtained by a RACEmethod using SMART RACE cDNA amplification kit (Clontech) as describedin Pastan I, Beers R, Bera T K. Recombinant immunotoxins in thetreatment of cancer. Methods Mol Biol 2004; 248:503-18, or other methodfor preparation of cDNA known to those of skill in the art. PreparedcDNAs are then used as the template for PCR reactions to amplify thedesired V_(H) and V_(L) fragments. The PCR products can then be clonedinto an appropriate vector, either directly into a vector containing thecytotoxin, or transferred to a shuttle vector, such as pCR4®-TOPO®,using the TOPO TA cloning kit (Invitrogen). The V_(H) and V_(L) chainscan then be assembled into a single chain Fv (scFv) and fused to acytotoxin as described in Pastan I, Beers R, Bera T K. “Recombinantimmunotoxins in the treatment of cancer.” Methods Mol Biol 2004;248:503-18, which is incorporated herein by reference in its entirety.As another example, Fab and F(ab′)₂ fragments of the invention may beproduced by proteolytic cleavage of immunoglobulin molecules, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments). F(ab′)₂ fragments contain the variable region, thelight chain constant region and the CH₁ domain of the heavy chain.

Further, the antibodies of the present invention can also be generatedusing various phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles that carry the polynucleotide sequences encoding them.In particular, DNA sequences encoding V_(H) and V_(L) domains areamplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of IL3Rα-expressing tissues). The DNA encoding the V_(H) andV_(L) domains are recombined together with an scFv linker by PCR andcloned into a phagemid vector. The vector is electroporated in E. coliand the E. coli is infected with helper phage. Phage used in thesemethods are typically filamentous phage including fd and M13 and theV_(H) and V_(L) domains are usually recombinantly fused to either thephage gene III or gene VIII. Phage expressing an antigen binding domainthat binds to a particular antigen can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Examples of phage display methods that can beused to make the antibodies of the present invention include thosedisclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ameset al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al.,1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18;Burton et al., 1994, Advances in Immunology 57:191-280; Internationalapplication No. PCT/GB91/01134; Griffiths et al., 1994, EMBO J13:3245-3260; Winter et al., 1994, Annu. Rev. Immunol. 12:433-455; Livet al., 2004, Cancer Res. 64:704-710; International publication Nos. WO90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceFab, Fab′ and F(ab′)₂ fragments can also be employed using methods knownin the art such as those disclosed in PCT publication No. WO 92/22324;Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995,AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043 (saidreferences incorporated by reference in their entireties).

To generate whole antibodies, PCR primers including V_(H) or V_(L)nucleotide sequences, a restriction site, and a flanking sequence toprotect the restriction site can be used to amplify the V_(H) or V_(L)sequences in scFv clones. Utilizing cloning techniques known to those ofskill in the art, the PCR amplified V_(H) domains can be cloned intovectors expressing a V_(H) constant region, e.g., the human gamma 4constant region, and the PCR amplified V_(L) domains can be cloned intovectors expressing a V_(L) constant region, e.g., human kappa or lambdaconstant regions. Preferably, the vectors for expressing the V_(H) orV_(L) domains comprise an EF-1α promoter, a secretion signal, a cloningsite for the variable domain, constant domains, and a selection markersuch as neomycin. The V_(H) and V_(L) domains may also cloned into onevector expressing the necessary constant regions. The heavy chainconversion vectors and light chain conversion vectors are thenco-transfected into cell lines to generate stable or transient celllines that express full-length antibodies, e.g., IgG, using techniquesknown to those of skill in the art.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use humanized antibodiesor chimeric antibodies. Completely human antibodies and humanizedantibodies are particularly desirable for therapeutic treatment of humansubjects. Human antibodies can be made by a variety of methods known inthe art including phage display methods described above using antibodylibraries derived from human immunoglobulin sequences. See also U.S.Pat. Nos. 4,444,887 and 4,716,111; and International publication Nos. WO98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice that areincapable of expressing functional endogenous immunoglobulins, but thatcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then be bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, 1995, Int. Rev. Immunol. 13:65 93.

For a detailed discussion of methods for producing human antibodies andhuman monoclonal antibodies and protocols for producing such antibodies,see, e.g., Tomizuka et al., 2000 PNAS USA 97:722-727; Davis et al.,2004, Methods Mol. Biol. 248:191-200; Lagerkvist et al., 1995,Biotechniques 18:862-869; Babcook et al., 1996 PNAS USA 93:7843-7848;International publication Nos. WO 98/24893, WO 96/34096, and WO96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporatedby reference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Methodsfor producing chimeric antibodies are known in the art. See e.g.,Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214;Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat.Nos. 5,807,715, 4,816,567, 4,816,397, and 6,311,415, which areincorporated herein by reference in their entirety.

A humanized antibody is an antibody that is capable of binding to apredetermined antigen and that comprises a framework region havingsubstantially the amino acid sequence of a human immunoglobulin and aCDR having substantially the amino acid sequence of a non-humanimmunoglobulin. A humanized antibody comprises substantially all of atleast one, and typically two, variable domains (Fab, Fab′, F(ab′)2,Fabc, Fv), in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence. Preferably, a humanizedantibody also comprises at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. Ordinarily, theantibody will contain both the light chain as well as at least thevariable domain of a heavy chain. The antibody also may include the CH1,hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanizedantibody can be selected from any class of immunoglobulins, includingIgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2,IgG3, and IgG4. Usually the constant domain is a complement fixingconstant domain where it is desired that the humanized antibody exhibitcytotoxic activity, and the class is typically IgG1. Where suchcytotoxic activity is not desirable, the constant domain may be of theIgG2 class. The humanized antibody may comprise sequences from more thanone class or isotype, and selecting particular constant domains tooptimize desired effector functions is within the ordinary skill in theart. The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor CDR orthe consensus framework may be mutagenized by substitution, insertion ordeletion of at least one residue so that the CDR or framework residue atthat site does not correspond to either the consensus or the importantibody. Such mutations, however, will not be extensive. Usually, atleast 75% of the humanized antibody residues will correspond to those ofthe parental framework and CDR sequences, more often 90%, and mostpreferably greater than 95%. A humanized antibody can be produced usingvariety of techniques known in the art, including but not limited to,CDR-grafting (see e.g., European Patent No. EP 239,400; InternationalPublication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101,and 5,585,089, each of which is incorporated herein in its entirety byreference), veneering or resurfacing (see e.g., European Patent Nos. EP592,106 and EP 519,596; Padlan, 1991, Molecular Immunology28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973, each of whichis incorporated herein by its entirety by reference), chain shuffling(see e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in itsentirety by reference), and techniques disclosed in, e.g., U.S. Pat.Nos. 6,407,213, 5,766,886, International Publication No. WO 9317105, Tanet al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng.13(5):353 60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca etal., J. Biol. Chem. 272(16):10678 84 (1997), Roguska et al., ProteinEng. 9(10):895 904 (1996), Couto et al., Cancer Res. 55 (23Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717 22(1995), Sandhu J S, Gene 150(2):409 10 (1994), and Pedersen et al., J.Mol. Biol. 235(3):959 73 (1994), each of which is incorporated herein inits entirety by reference. Often, framework residues in the frameworkregions will be substituted with the corresponding residue from the CDRdonor antibody to alter, preferably improve, antigen binding. Theseframework substitutions are identified by methods well known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature 332:323, which are incorporated herein byreference in their entireties.)

Diabodies, triabodies, and tetrabodies can be produced by techniquesknown to one of skill in the art. See, e.g., Kipriyanov, 2002, MethodsMol. Biol. 178:317-331; Todorovska et al., 2001 J. Immunol. Methods248:47-66; and Poljak et al., 1994, Structure 2:1121-1123, each of whichare incorporated herein by reference in their entirety, for methods forproducing diabodies, triabodies, and tetrabodies. Single domainantibodies can also be produced by techniques known to one of skill inthe art. For a description of techniques to produce single domainantibodies, see, e.g., Holliger & Hudson, 2005 Nat. Biotechnol.23:1126-1136, Riechmann et al., 1999, J. Immunol. Methods 231:25-38; andDick, 1990, BMJ 300:659-600, each of which is incorporated herein byreference in its entirety.

Generation of intrabodies is well-known to the skilled artisan and isdescribed, for example, in U.S. Pat. Nos. 6,004,940; 6,072,036;5,965,371, which are incorporated by reference in their entiretiesherein. Further, the construction of intrabodies is discussed in Ohageand Steipe, 1999, J. Mol. Biol. 291:1119-1128; Ohage et al., 1999, J.Mol. Biol. 291:1129-1134; and Wirtz and Steipe, 1999, Protein Science8:2245-2250, which references are incorporated herein by reference intheir entireties. Recombinant molecular biological techniques such asthose described for recombinant production of antibodies may also beused in the generation of intrabodies.

5.6.1 Nucleotide Sequences Encoding an Antibody

The invention provides nucleic acid sequences comprising a nucleotidesequence encoding an antibody or an antibody conjugate that binds toIL3Rα. In a specific embodiment, such nucleic acid sequences areisolated. The invention also encompasses nucleic acid sequences thathybridize under high, intermediate, or lower stringency hybridizationconditions, e.g., as defined supra, to nucleic acid sequences thatencode an antibody of the invention.

The nucleic acid sequence may be obtained, and the nucleotide sequenceof the nucleic acid sequence determined, by any method known in the art.The nucleotide sequence of antibodies specific for IL3Rα or a desiredepitope on IL3Rα can be obtained, e.g., from the literature or adatabase such as GenBank. Such a nucleic acid sequence encoding theantibody may be assembled from chemically synthesized oligonucleotides(e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242),which, briefly, involves the synthesis of overlapping oligonucleotidescontaining portions of the sequence encoding the antibody, annealing andligating of those oligonucleotides, and then amplification of theligated oligonucleotides by PCR. FIGS. 1 to 4 and Table 1 provide thenucleotide and/or amino acid sequences of the V_(H) domains, V_(L)domains and CDRs of the 26929, 32703, 32701 and 32716 antibodies.

Alternatively, a nucleic acid sequence encoding an IL3Rα antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid—preferably polyA+RNA—isolated from, any tissue or cells expressing the antibody, suchas hybridoma cells selected to express an antibody of the invention) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence to identify, e.g., a cDNAclone from a cDNA library that encodes the antibody. Amplified nucleicacids generated by PCR may then be cloned into replicable cloningvectors using any method well known in the art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., 1990,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998,Current Protocols in Molecular Biology, John Wiley & Sons, NY, which areboth incorporated by reference herein in their entireties), to generateantibodies having a different amino acid sequence, for example, tocreate amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, one or more of the CDRs is inserted withinframework regions using routine recombinant DNA techniques. Theframework regions may be naturally occurring or consensus frameworkregions, and preferably human framework regions (see, e.g., Chothia etal., 1998, J. Mol. Biol. 278: 457-479 for a listing of human frameworkregions). Preferably, the nucleic acid sequence generated by thecombination of the framework regions and CDRs encodes an antibody thatbinds to the IL3Rα chain. In a specific embodiment, one or more aminoacid substitutions may be made within the framework regions, and incertain embodiments, the amino acid substitutions improve binding of theantibody to its antigen. Additionally, such methods may be used to makeamino acid substitutions or deletions of one or more variable regioncysteine residues participating in an intrachain disulfide bond togenerate antibody molecules lacking one or more intrachain disulfidebonds. Other alterations to the nucleic acid sequence are encompassed bythe present invention and within the skill of the art.

5.6.2 Recombinant Expression of an Antibody

Recombinant expression of an antibody that binds to IL3Rαdescribedabove, requires construction of an expression vector containing anucleic acid sequence that encodes the antibody. Once a nucleic acidsequence encoding an antibody molecule, heavy or light chain of anantibody, or fragment thereof (preferably, but not necessarily,containing the heavy or light chain variable domain) of the inventionhas been obtained, the vector for the production of the antibodymolecule may be produced by recombinant DNA technology using techniqueswell-known in the art. Thus, methods for preparing an antibody byexpressing a nucleic acid sequence encoding the antibody are describedherein. Methods that are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, a heavy orlight chain of an antibody, a heavy or light chain variable domain of anantibody or a fragment thereof, or a heavy or light chain CDR, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,International Publication No. WO 86/05807; International Publication No.WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy chain, the entire light chain, or both the entire heavy and lightchains.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a nucleic acid sequence encoding anantibody, or a heavy or light chain thereof, or fragment thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention (see, e.g., U.S. Pat. No.5,807,715). Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced and subsequently purified,but also represent cells that may, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibodymolecule of the invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces cerevisiae or Pichia pastoris) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). Preferably, bacterial cells such as Escherichiacoli, and more preferably, eukaryotic cells, especially for theexpression of whole recombinant antibody molecules, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus, is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990,Bio/Technology 8:2). In a specific embodiment, the expression ofnucleotide sequences encoding antibodies that bind to the IL3R alphachain is regulated by a constitutive promoter, inducible promoter, ortissue specific promoter.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors that direct the expressionof high levels of fusion protein products that are readily purified maybe desirable. Such vectors include, but are not limited to, the E. coliexpression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in whichthe antibody coding sequence may be ligated individually into the vectorin frame with the lac Z coding region so that a fusion protein isproduced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509);and the like. pGEX vectors may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption and binding to matrix glutathione agarosebeads followed by elution in the presence of free glutathione. The pGEXvectors are designed to include thrombin or factor Xa protease cleavagesites so that the cloned target gene product can be released from theGST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of virus-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted into the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing the antibody molecule in infected hosts(e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359).Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see, e.g., Bittneret al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK,293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O, and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theantibody molecule may be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.) and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci, which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines that express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used; as non-limiting examples, theherpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wuand Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan andAnderson, 1993, Ann. Rev. Biochem. 62: 191-217; May, 1993, TIB TECH11(5):155-2 15); and hygro, which confers resistance to hygromycin(Santerre et al., 1984, Gene 30:147). Methods commonly known in the artof recombinant DNA technology may be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, N Y (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, N Y (1990); and in Chapters 12 and13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, JohnWiley & Sons, N Y (1994); Colberre-Garapin et al., 1981, J. Mol. Biol.150:1, which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler,1980, Proc. Natl. Acad. Sci. USA 77:2 197). The coding sequences for theheavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies may be fused to heterologous polypeptide sequences describedherein or otherwise known in the art to facilitate purification.

Once a conjugate of the invention has been produced by recombinantexpression or by chemical synthesis, it may be purified by any methodknown in the art for purification of a protein, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

5.7 Pharmaceutical Compositions and Routes of Administration

The present invention provides compositions comprising an antibody orantibody conjugate of the invention and carrier. In one embodiment, theinvention provides compositions comprising an antibody of the invention.In a another embodiment, the invention provides compositions comprisingan antibody conjugate, such as an immunotoxin.

The invention provides a pharmaceutical composition comprising aneffective amount of an antibody or antibody conjugate of the inventionand a pharmaceutically acceptable carrier or vehicle. In a specificembodiment, a pharmaceutical composition comprises an effective amountof an antibody of the invention and a pharmaceutical acceptable carrieror vehicle. In another embodiment, a pharmaceutical compositioncomprises an effective amount of an antibody conjugate of the inventionand a pharmaceutically acceptable carrier or vehicle. The pharmaceuticalcompositions are suitable for veterinary and/or human administration.

The pharmaceutical compositions of the present invention can be in anyform that allows for the composition to be administered to a subject,said subject preferably being an animal, including, but not limited to ahuman, mammal, or non-human animal, such as a cow, horse, sheep, pig,fowl, cat, dog, mouse, rat, rabbit, guinea pig, etc., and is morepreferably a mammal, and most preferably a human.

The compositions of the invention can be in the form of a solid, liquidor gas (aerosol). Typical routes of administration may include, withoutlimitation, oral, topical, parenteral, sublingual, rectal, vaginal,ocular, intradermal, intratumoral, intracerebral, intrathecal, andintranasal. Parenteral administration includes subcutaneous injections,intravenous, intramuscular, intraperitoneal, intrapleural, intrasternalinjection or infusion techniques. In a specific embodiment, thecompositions are administered parenterally. In a more specificembodiment, the compositions are administered intravenously.Pharmaceutical compositions of the invention can be formulated so as toallow an antibody or antibody conjugate of the invention to bebioavailable upon administration of the composition to a subject.Compositions can take the form of one or more dosage units, where, forexample, a tablet can be a single dosage unit, and a container of anantibody or antibody conjugate of the invention in aerosol form can holda plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of subject (e.g.,human), the overall health of the subject, the type of cancer thesubject is in need of treatment of, the use of the composition as partof a multi-drug regimen, the particular form of the antibody or antibodyconjugate of the invention, the manner of administration, and thecomposition employed.

The pharmaceutically acceptable carrier or vehicle may be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) can begaseous, so as to provide an aerosol composition useful in, e.g.,inhalatory administration.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich a conjugate of the invention is administered. Such pharmaceuticalcarriers can be liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. The carriers can besaline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea, and the like. In addition, auxiliary, stabilizing,thickening, lubricating and coloring agents can be used. In oneembodiment, when administered to a subject, the conjugates of theinvention and pharmaceutically acceptable carriers are sterile. Water isa preferred carrier when the conjugate of the invention is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present compositions, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

The composition may be intended for oral administration, and if so, thecomposition is preferably in solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as ethyl cellulose,carboxymethylcellulose, microcrystalline cellulose, or gelatin;excipients such as starch, lactose or dextrins, disintegrating agentssuch as alginic acid, sodium alginate, Primogel, corn starch and thelike; lubricants such as magnesium stearate or Sterotex; glidants suchas colloidal silicon dioxide; sweetening agents such as sucrose orsaccharin, a flavoring agent such as peppermint, methyl salicylate ororange flavoring, and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, e.g., agelatin capsule, it can contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol, cyclodextrin or afatty oil.

The pharmaceutical composition can be in the form of a liquid, e.g., anelixir, syrup, solution, emulsion or suspension. The liquid can beuseful for oral administration or for delivery by injection. Whenintended for oral administration, a composition can comprise one or moreof a sweetening agent, preservatives, dye/colorant and flavor enhancer.In a composition for administration by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent can also be included.

The liquid compositions of the invention, whether they are solutions,suspensions or other like form, can also include one or more of thefollowing: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or digylcerides whichcan serve as the solvent or suspending medium, polyethylene glycols,glycerin, cyclodextrin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as EDTA; buffers such as acetates, citrates or phosphates andagents for the adjustment of tonicity such as sodium chloride ordextrose. A parenteral composition can be enclosed in an ampoule, adisposable syringe or a multiple-dose vial made of glass, plastic orother material. Physiological saline is a preferred adjuvant. Aninjectable composition is preferably sterile.

The pharmaceutical compositions comprise an effective amount of anantibody or antibody conjugate of the invention such that a suitabledosage will be obtained (see infra, for suitable dosages). Typically,this amount is at least 0.01% of an antibody or antibody conjugate ofthe invention by weight of the composition. When intended for oraladministration, this amount can be varied to be between 0.1% and 80% byweight of the composition. Preferred oral compositions can comprise frombetween 4% and 50% of the antibody or antibody conjugate of theinvention by weight of the composition. Preferred compositions of thepresent invention are prepared so that a parenteral dosage unit containsfrom between 0.01% and 2% by weight of the antibody or antibodyconjugate of the invention.

The compositions of the invention can be administered by any convenientroute, for example, by infusion or bolus injection, by absorptionthrough epithelial or mucocutaneous linings (e.g., oral mucosa, rectaland intestinal mucosa, etc.). Administration can be systemic or local.Various delivery systems are known, e.g., microparticles, microcapsules,capsules, etc., and may be useful for administering an antibody orantibody conjugate of the invention. In certain embodiments, more thanone antibody or antibody conjugate of the invention is administered to asubject. Methods of administration may include, but are not limited to,oral administration and parenteral administration; parenteraladministration including, but not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous; intranasal,epidural, sublingual, intranasal, intracerebral, intraventricular,intrathecal, intravaginal, transdermal, rectally, by inhalation, ortopically to the ears, nose, eyes, or skin. The preferred mode ofadministration is left to the discretion of the practitioner, and willdepend in-part upon the site of the medical condition (such as the siteof cancer, a cancerous tumor or a precancerous condition).

In one embodiment, the antibodies or antibody conjugates of theinvention are administered parenterally. In a specific embodiment, theantibodies or antibody conjugates of the invention are administeredintravenously. In another embodiment, the antibodies or antibodyconjugates of the invention are administered by continuous infusion. Ina particular embodiment, the antibodies or antibody conjugates of theinvention are administered by an infusion that lasts for about 15minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1hour, or about 2 hours.

In specific embodiments, it can be desirable to administer one or moreantibodies or antibody conjugates of the invention locally to the areain need of treatment.

This can be achieved, for example, and not by way of limitation, bylocal infusion during surgery; topical application, e.g., in conjunctionwith a wound dressing after surgery; by injection; by means of acatheter; by means of a suppository; or by means of an implant, theimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as silastic membranes, or fibers. In one embodiment,administration can be by direct injection at the site (or former site)of a cancer, tumor, or precancerous tissue. In certain embodiments, itcan be desirable to introduce one or more antibodies or antibodyconjugates of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection.Intraventricular injection can be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. In certain embodiments, one or more compounds of theinvention can be injected intraperitoneally.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the antibodies or antibody conjugates of theinvention can be formulated as a suppository, with traditional bindersand carriers such as triglycerides.

In yet another embodiment, the antibody or antibody conjugates of theinvention can be delivered in a controlled release system. In oneembodiment, a pump can be used (see Sefton, CRC Crit. Ref. Biomed. Eng.1987, 14, 201; Buchwald et al., Surgery 1980, 88: 507; Saudek et al., NEngl. J. Med. 1989, 321: 574). In another embodiment, polymericmaterials can be used (see Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. 1983, 23, 61; see also Levy et al., Science 1985,228, 190; During et al., Ann. Neurol., 1989, 25, 351; Howard et al., J.Neurosurg., 1989, 71, 105). In yet another embodiment, acontrolled-release system can be placed in proximity of the target ofthe antibodies or antibody conjugates of the invention, e.g., the brain,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,in Medical Applications of Controlled Release, supra, vol. 2, 1984, pp.115-138). Other controlled-release systems discussed in the review byLanger (Science 1990, 249, 1527-1533) can be used.

In another embodiment, polymeric materials can be used to achievecontrolled or sustained release of the antibodies or antibody conjugatesof the invention (see, e.g., U.S. Pat. Nos. 5,679,377; 5,916,597;5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99/15154; andPCT Publication No. WO 99/20253. Examples of polymers used in sustainedrelease formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferredembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. In one embodiment, the pharmaceutically acceptable carrier is acapsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences by E. W. Martin.

Sustained or directed release compositions that can be formulatedinclude, but are not limited to, antibodies or antibody conjugates ofthe invention protected with differentially degradable coatings, e.g.,by microencapsulation, multiple coatings, etc. It is also possible tofreeze-dry the compositions and use the lyophilizates obtained, forexample, for the preparation of products for injection.

In a preferred embodiment, the antibodies or antibody conjugates of theinvention are formulated in accordance with routine procedures as apharmaceutical composition adapted for intravenous administration toanimals, particularly human beings. Typically, the carriers or vehiclesfor intravenous administration are sterile isotonic aqueous buffersolutions. Where necessary, the compositions can also include asolubilizing agent. Compositions for intravenous administration canoptionally comprise a local anesthetics such as lignocaine to ease painat the site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachet indicating the quantity ofactive agent. Where an antibody or antibody conjugate of the inventionis to be administered by infusion, it can be dispensed, for example,with an infusion bottle containing sterile pharmaceutical grade water orsaline. Where the antibody or antibody conjugate of the invention isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

Compositions for oral delivery can be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs, for example. Orally administered compositions cancontain one or more optional agents, for example, sweetening agents suchas fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.Moreover, where in tablet or pill form, the compositions can be coatedto delay disintegration and absorption in the gastrointestinal tractthereby providing a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving complex are also suitable for orally administered compositionsof the invention. In these later platforms, fluid from the environmentsurrounding the capsule is imbibed by the driving complex, which swellsto displace the agent or agent composition through an aperture. Thesedelivery platforms can provide an essentially zero order deliveryprofile as opposed to the spiked profiles of immediate releaseformulations. A time-delay material such as glycerol monostearate orglycerol stearate can also be used. Oral compositions can includestandard carriers such as mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Such carriersare preferably of pharmaceutical grade.

The pharmaceutical compositions of the invention can be intended fortopical administration, in which case the carrier can be in the form ofa solution, emulsion, ointment or gel base. The base, for example, cancomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents can be present in acomposition for topical administration. If intended for transdermaladministration, the composition can be in the form of a transdermalpatch or an iontophoresis device. Topical formulations can comprise aconcentration of a conjugate of the invention of from between 0.01% and10% w/v (weight per unit volume of composition).

The compositions can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

The compositions can consist of gaseous dosage units, e.g., it can be inthe form of an aerosol. The term aerosol is used to denote a variety ofsystems ranging from those of colloidal nature to systems consisting ofpressurized packages. Delivery can be by a liquefied or compressed gasor by a suitable pump system that dispenses the active ingredients.Aerosols of the compositions can be delivered in single phase,bi-phasic, or tri-phasic systems in order to deliver the composition.Delivery of the aerosol includes the necessary container, activators,valves, subcontainers, spacers and the like, which together can form akit. Preferred aerosols can be determined by one skilled in the art,without undue experimentation.

Whether in solid, liquid or gaseous form, the compositions of thepresent invention can comprise an additional active agent selected fromamong those including, but not limited to, an additional prophylacticagent, an additional therapeutic agent, an antiemetic agent, ahematopoietic colony stimulating factor, an adjuvant therapy, a vaccineor other immune stimulating agent, an antibody/antibody fragment-basedagent, an anti-depressant and an analgesic agent. For instance in aparticular embodiment, the pharmaceutical composition comprises aconjugate of the invention, an additional agent, and a pharmaceuticallyacceptable carrier or vehicle.

The pharmaceutical compositions can be prepared using methodology wellknown in the pharmaceutical art. For example, a composition intended tobe administered by injection can be prepared by combining a conjugate ofthe invention with water so as to form a solution. A surfactant can beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants are complexes that can non-covalently interactwith a conjugate of the invention so as to facilitate dissolution orhomogeneous suspension of the conjugate of the invention in the aqueousdelivery system.

In one embodiment, the pharmaceutical compositions of the presentinvention may comprise one or more other therapies.

The present invention provides pharmaceutical compositions comprising anantibody of the invention in an amount effective to reduce a cancer stemcell population and/or cancer cell population in an animal with oranimal model for myeloid leukemia or another cancer associated withIL3Rα-expressing cells by about 25%, 30%, 40%, 50%, 75%, 80%, 85%, 90%,95% or 98% relative to a negative control. In a specific embodiment, thereduction in the cancer stem cell population in the animal or animalmodel is at least 25% relative to a negative control. In someembodiments, the animal is a human. In other embodiments, the animal isa non-human animal.

The present invention provides pharmaceutical compositions comprising anantibody conjugate in an amount effective to reduce a cancer stem cellpopulation and/or cancer cell population in an animal with or animalmodel for myeloid leukemia or another cancer associated withIL3Rα-expressing cells by about 25%, 30%, 40%, 50%, 75%, 80%, 85%, 90%,95% or 98% relative to a negative control, wherein the conjugatecomprises an antibody that binds to the IL3Rα chain and a cytotoxicagent or other anticellular moiety. In a specific embodiment, theconjugate comprises V_(H) and V_(L) domains of the 26292, 32701, 32701or 32716 antibody. In another embodiment, the conjugate comprises one ormore or all of the CDRs of the CDRs of the 26292, 32703, 32701 or 32716antibody. In a specific embodiment, the reduction in the cancer stemcell population in the animal or animal model is at least 25% relativeto a negative control. In some embodiments, the animal is human. Inother embodiments, the animal is a non-human animal.

5.8 Therapeutic and Prophylactic Uses of Conjugates

The present invention provides methods of treating, preventing and/ormanaging a disorder characterized by cells expressing the IL-3 receptoralpha subunit, the methods comprising administering to a subject(preferably, a human) in need thereof a pharmaceutical compositioncomprising an effective amount of an antibody or antibody conjugate ofthe invention. In one embodiment, the antibody or antibody conjugates ofthe invention are administered as monotherapy for the prevention,treatment, and/or management of a disorder characterized by cellsexpressing the IL-3 receptor alpha subunit. In other embodiments, theantibody or antibody conjugates are administered in combination withanother therapy. In certain embodiments, the antibodies and antibodyconjugates of the invention are administered in combination, andoptionally with other therapies.

The present invention is directed to therapies which involveadministering one or more of the antibody or antibody conjugates of theinvention and compositions comprising the antibody or antibodyconjugates to a subject, preferably a human subject, for preventing,treating, managing, and/or ameliorating disease or disorder thatdisplays or is characterized by IL-3 receptor alpha subunit expressionor one or more symptoms thereof. In one embodiment, the inventionprovides a method of preventing, treating, managing, and/or amelioratinga disease or disorder that displays or is characterized by IL-3 receptoralpha subunit expression or one or more symptoms thereof, said methodcomprising administering to a subject in need thereof an effectiveamount of one or more antibody or antibody conjugates of the invention.Such diseases and disorders include cancer, allergic diseases,inflammatory diseases, and autoimmune diseases.

The invention also provides methods comprising administering to asubject in need thereof an antibody or antibody conjugate of theinvention and one or more therapies (e.g., one or more prophylactic ortherapeutic agents) other than the antibody or antibody conjugate of theinvention that are currently being used, have been used, are known to beuseful, or may be useful in the prevention, treatment, management,and/or amelioration of a disease or disorder that displays or ischaracterized by IL-3 receptor alpha subunit expression or one or moresymptoms thereof. The prophylactic or therapeutic agents of thecombination therapies of the invention can be administered sequentiallyor concurrently. In a specific embodiment, the combination therapies ofthe invention comprise an effective amount of an antibody or antibodyconjugate of the invention and an effective amount of at least one othertherapy which has the same mechanism of action as said an antibody orantibody conjugate. In a specific embodiment, the combination therapiesof the invention comprise an effective amount of an antibody or antibodyconjugate of the invention and an effective amount of at least one othertherapy (e.g., prophylactic or therapeutic agent) which has a differentmechanism of action than said antibody or antibody conjugate. In certainembodiments, the combination therapies of the present invention improvethe prophylactic or therapeutic effect of an antibody or antibodyconjugate of the invention by functioning together with an antibody orantibody conjugate to have an additive or synergistic effect. In certainembodiments, the combination therapies of the present invention reducethe side effects associated with the prophylactic or therapeutic agents.In other embodiments, the combination therapies are administered priorto, during, or after the administration of the compositions of theinvention.

Cancer or a neoplastic disease, including, but not limited to,neoplasms, tumors, metastases, or any disease or disorder characterizedby uncontrolled cell growth, can be treated, suppressed, delayed,managed, inhibited or prevented by administering to a subject in needthereof a prophylactically effective regimen or a therapeuticallyeffective regimen, the regimen comprising administering to the patient acompound of the invention. In specific embodiments, the inventionencompasses the treatment, suppression, delaying, management, inhibitingof growth and/or progression, and prevention of cancer or neoplasticdisease as described herein.

In one embodiment, the antibody or antibody conjugates of the inventionare administered as monotherapy for the prevention, treatment, and/ormanagement of cancer.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient an antibody or antibody conjugate of theinvention or a pharmaceutical composition of the invention, wherein thepatient has been diagnosed with cancer.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient an antibody or antibody conjugate of theinvention or a pharmaceutical composition of the invention, wherein thepatient has relapsed from cancer.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient an antibody or antibody conjugate of theinvention or a pharmaceutical composition of the invention, wherein thepatient has failed or is failing therapy.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient an antibody or antibody conjugate of theinvention or a pharmaceutical composition of the invention, wherein thepatient is in remission from cancer.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient an antibody or antibody conjugate of theinvention or a pharmaceutical composition of the invention, wherein thepatient is refractory to therapy.

In one embodiment, the cancer is a hematologic cancer. For instance, thecancer can be leukemia, lymphoma, myelodysplastic syndrome (MDS), ormyeloma. In another embodiment, the cancer is a solid tumor.

In one embodiment of this aspect, the patient has received or isreceiving another therapy. In another embodiment of this aspect, thepatient has not previously received a therapy for the prevention,treatment, and/or management of the cancer.

The medical practitioner can diagnose the patient using any of theconventional cancer screening methods including, but not limited tophysical examination (e.g., prostate examination, rectal examination,breast examination, lymph nodes examination, abdominal examination, skinsurveillance, testicular exam, general palpation), visual methods (e.g.,colonoscopy, bronchoscopy, endoscopy), PAP smear analyses (cervicalcancer), stool guaiac analyses, blood tests (e.g., complete blood count(CBC) test, prostate specific antigen (PSA) test, carcinoembryonicantigen (CEA) test, cancer antigen (CA)-125 test, alpha-fetoprotein(AFP), liver function tests), karyotyping analyses, bone marrow analyses(e.g., in cases of hematological malignancies), histology, cytology,flow cytometry, a sputum analysis, and imaging methods (e.g., computedtomography (CT), magnetic resonance imaging (MRI), ultrasound, X-rayimaging, mammography, PET scans, bone scans, radionuclide scans).

Another aspect of the invention relates to a method of preventing,treating, and/or managing a solid tumor in a patient (e.g., a humanpatient), the method comprising administering to a patient in needthereof a prophylactically effective regimen or a therapeuticallyeffective regimen, the regimen comprising administering to the patientan antibody or antibody conjugate or pharmaceutical composition of theinvention wherein the patient has been diagnosed with a solid tumor, andwherein the patient has undergone a primary therapy to reduce the bulkof the tumor. The primary therapy to reduce the tumor bulk size ispreferably a therapy other than an antibody or antibody conjugate of theinvention. In specific embodiment of this aspect, the solid tumor isfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer,kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovariancancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer,nasal cancer, throat cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular cancer, small cell lung carcinoma, bladder carcinoma,lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, or retinoblastoma.

Another aspect of the invention relates to a method of preventing,treating, and/or managing cancer, the method comprising administering toa patient in need thereof a prophylactically effective regimen or atherapeutically effective regimen, the regimen comprising administeringto the patient an antibody or antibody conjugate of the invention,wherein the patient received another therapy. In some embodiments, theprior therapy is, for example, chemotherapy, small molecule therapy,radioimmunotherapy, toxin therapy, prodrug-activating enzyme therapy,biologic therapy, antibody therapy, surgical therapy, hormone therapy,immunotherapy, anti-angiogenic therapy, targeted therapy, epigenetictherapy, demethylation therapy, histone deacetylase inhibitor therapy,differentiation therapy, radiation therapy, or any combination thereof.

In some embodiments, the prior therapy has failed in the patient. Insome embodiments, the therapeutically effective regimen comprisingadministration of an antibody or antibody conjugate of the invention isadministered to the patient immediately after the patient has undergonethe prior therapy. For instance, in certain embodiments, the outcome ofthe prior therapy may be unknown before the patient is administered theantibody or antibody conjugate.

Another aspect of the invention relates to a method of preventing cancerin a patient (e.g., a human patient), the method comprisingadministering to a patient in need thereof a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient an antibody or antibody conjugate of theinvention, wherein the cancer in the patient has entered remission. Insome embodiments of this aspect, through administration of aprophylactically effective regimen or a therapeutically effectiveregimen, the medical practitioner can effectively cure the cancer, orprevent its reoccurrence.

Another aspect of the invention relates to a method of preventing,treating, and/or managing cancer in a patient (e.g., a human patient),the method comprising administering to a patient in need thereof aprophylactically effective regimen or a therapeutically effectiveregimen, the regimen comprising administering to the patient a compoundor composition of the invention, wherein the antibody or antibodyconjugate is administered at a dose that is lower than the maximumtolerated dose (MTD) over a period of three months, four months, sixmonths, nine months, 1 year, 2 years, 3 years, 4 years, or more.

Another aspect of the invention relates to a method of preventing,treating, and/or managing cancer in a patient (e.g., a human patient),the method comprising administering to a patient in need thereof aprophylactically effective regimen or a therapeutically effectiveregimen, the regimen comprising administering to the patient an antibodyor antibody conjugate of the invention, wherein the antibody or antibodyconjugate is administered at a dose that is lower than the humanequivalent dosage (HED) of the no observed adverse effect level (NOAEL)over a period of three months, four months, six months, nine months, 1year, 2 years, 3 years, 4 years, or more. The NOAEL, as determined inanimal studies, is useful in determining the maximum recommendedstarting dose for human clinical trials. For instance, the NOAELs can beextrapolated to determine human equivalent dosages. Typically, suchextrapolations between species are conducted based on the doses that arenormalized to body surface area (i.e., mg/m²). In specific embodiments,the NOAELs are determined in mice, hamsters, rats, ferrets, guinea pigs,rabbits, dogs, primates (monkeys, marmosets, squirrel monkeys, baboons),micropigs, or minipigs. For a discussion on the use of NOAELs and theirextrapolation to determine human equivalent doses, see Guidance forIndustry Estimating the Maximum Safe Starting Dose in Initial ClinicalTrials for Therapeutics in Adult Healthy Volunteers, U.S. Department ofHealth and Human Services Food and Drug Administration Center for DrugEvaluation and Research (CDER), Pharmacology and Toxicology, July 2005.

While not being bound by any specific theory, Applicants believe that bythe administration of the prophylactically and/or therapeuticallyeffective regimens, the cancer stem cell population of a cancer/tumor isstabilized or reduced, so as to limit or prevent the potentialrepopulation of the tumor.

In certain embodiments of these aspects, the regimens compriseadministering a prophylactically effective regimen and/or atherapeutically effective regimen, wherein the regimen results in areduction in the cancer stem cell population in the patient. In oneembodiment, the patient undergoing the regimen is monitored to determinewhether the regimen has resulted in a reduction in the cancer stem cellpopulation in the patient.

Typically, the monitoring of the amount of cancer stem cells isconducted by detecting the amount of cancer stem cells in a specimenextracted from the patient. Methods of detecting the amount of cancerstem cells in a specimen are described infra in Section 5.9. Thismonitoring step is typically performed at least 1, 2, 4, 6, 7, 8, 10,12, 14, 15, 16, 18, 20, or 30, 60, 90, 120 days, 6 months, 9 months, 12months, or >12 months after the patient begins receiving the regimen.

In some embodiments, the specimen may be a blood specimen, wherein theamount of cancer stem cells per unit of volume (e.g., 1 ml) or othermeasured unit (e.g., per unit field in the case of a histologicalanalysis) is quantitated. In certain embodiments, the amount of cancerstem cells is determined as a portion (e.g., a percentage) of the cancercells present in the blood specimen, as a subset of the cancer cellspresent in the blood specimen, or as a subset of a subset of the cancercells present in the blood specimen. The amount of cancer stem cells, inother embodiments, can be determined as a percentage of the total bloodcells.

In other embodiments, the specimen extracted from the patient is atissue specimen (e.g., a biopsy extracted from suspected canceroustissue), where the amount of cancer stem cells can be measured, forexample, on the basis of the amount of cancer stem cells per unit weightof the tissue. In certain embodiments, the amount of cancer stem cellsis determined as a portion (e.g., a percentage) of the cancer cellspresent in the tissue, as a subset of the cancer cells present in thetissue, or as a subset of a subset of the cancer cells present in thetissue.

The amount of cancer stem cells in the extracted specimen can becompared with the amount of cancer stem cells measured in referencesamples to assess the efficacy of the regimen, and the amelioration ofthe cancer under therapy. In one embodiment, the reference sample is aspecimen extracted from the patient undergoing therapy, wherein thespecimen is extracted from the patient at an earlier time point (e.g.,prior to receiving the regimen, as a baseline reference sample, or at anearlier time point while receiving the therapy). In another embodiment,the reference sample is extracted from a healthy, noncancer-afflictedpatient.

In other embodiments the amount of cancer stem cells in the extractedspecimen can be compared with a predetermined reference range. In aspecific embodiment, the predetermined reference range is based on i)the amount of cancer stem cells obtained from a population(s) ofpatients suffering from the same type of cancer as the patientundergoing the therapy, or ii) the amount of stem cells obtained from apopulation(s) of patients without cancer.

If the reduction in the amount of cancer stem cells is determined to betoo small upon comparing the amount of cancer stem cells in the specimenextracted from the patient undergoing the regimen with the referencespecimen, then the medical practitioner has a number of options toadjust the regimen. For instance, the medical practitioner can thenincrease either the dosage of the compound or composition of theinvention administered, the frequency of the administration, theduration of administration, or any combination thereof. In a specificembodiment, after the determination is made, a second effective amountof a compound or composition of the invention can be administered to thepatient.

In certain embodiments, if the reduction in the amount of cancer stemcells is determined to be acceptable upon comparing the amount of cancerstem cells in the sample obtained from the patient undergoing thetherapeutic or prophylactic regimen with the reference sample, then themedical practitioner may elect not to adjust the regimen. For instance,the medical practitioner may elect not to increase either the dosage ofthe compound or composition of the invention being administered, thefrequency of the administration, the duration of administration, or anycombination thereof. Further, the medical practitioner may elect to addadditional therapies or combine therapies.

In other embodiments, the regimens comprise administering aprophylactically effective regimen and/or a therapeutically effectiveregimen, wherein the regimen results in a reduction in the amount ofcancer cells in the patient. In one embodiment, the patient undergoingthe regimen is monitored to determine whether the regimen has resultedin a reduction in the amount of cancer cells in the patient.

Typically, the monitoring of the amount of cancer cells is conducted bydetecting the amount of cancer cells in a specimen extracted from thepatient. Methods of detecting the amount of cancer cells in a specimenare described infra in Section 5.10. This monitoring step is typicallyperformed at least 1, 2, 4, 6, 7, 8, 10, 12, 14, 15, 16, 18, 20, or 30,60, 90, 120 days, 6 months, 9 months, 12 months, or >12 months after thepatient begins receiving the regimen.

In some embodiments, the specimen may be a blood specimen, wherein theamount of cancer cells per unit of volume (e.g., 1 ml) or other measuredunit (e.g., per unit field in the case of a histological analysis) isquantitated. The cancer cell population, in certain embodiments, can bedetermined as a percentage of the total blood cells.

In some embodiments, the sample obtained from the patient may be a bonemarrow specimen, wherein the amount of cancer cells per unit of volume(e.g., 1 ml) or other measured unit (e.g., per unit field in the case ofa histological analysis) is quantitated. The cancer cell population, incertain embodiments, can be determined as a percentage of the total bonemarrow cells.

In other embodiments, the specimen extracted from the patient is atissue specimen (e.g., a biopsy extracted from suspected canceroustissue), where the amount of cancer cells can be measured, for example,on the basis of the amount of cancer cells per unit weight of thetissue. The amount of cancer cells can also be measured usingimmunohistochemistry or flow cytometry.

The amount of cancer cells in the extracted specimen can be comparedwith the amount of cancer cells measured in reference samples to assessthe efficacy of the regimen and amelioration of the cancer undertherapy. In one embodiment, the reference sample is a specimen extractedfrom the patient undergoing therapy, wherein the specimen from thepatient is extracted at an earlier time point (e.g., prior to receivingthe regimen, as a baseline reference sample, or at an earlier time pointwhile receiving the therapy). In another embodiment, the referencesample is extracted from a healthy, noncancer-afflicted patient.

In other embodiments the cancer cell population in the extractedspecimen can be compared with a predetermined reference range. In aspecific embodiment, the predetermined reference range is based on theamount of cancer cells obtained from a population(s) of patientssuffering from the same type of cancer as the patient undergoing thetherapy.

If the reduction in the cancer cell population is judged too small uponcomparing the amount of cancer cells in the specimen extracted from thepatients undergoing therapy with the reference specimen, then themedical practitioner has a number of options to adjust the therapeuticregimen. For instance, the medical practitioner can then either increasethe dosage of the compound or composition of the invention administered,the frequency of the administration, the duration of administration, orany combination thereof. In a specific embodiment, after thedetermination is made, a second effective amount of a compound orcomposition of the invention can be administered to the patient.

If the reduction in the cancer cell population is judged to be adequateupon comparing the amount of cancer cells in the specimen extracted fromthe patients undergoing therapy with the reference specimen, then themedical practitioner may elect not to adjust the therapeutic regimen.For instance, the medical practitioner may elect not to increase thedosage of the compound or composition of the invention administered, thefrequency of the administration, the duration of administration, or anycombination thereof.

The above monitoring methods can also be used to monitor the amount ofIL-3 receptor alpha subunit-expressing cells where the disease ordisorder is not a cancer, i.e., in allergic disease or autoimmunedisease.

In embodiments, the medical practitioner may elect to measure the cancerpopulation using in vivo imaging techniques. For example, a ligand for atumor marker can be conjugated to a radioisotope, photon emittingcompound, or other signal emitting compound, and then the ligand can beinjected into the patient. The cancer cells can then be quantitated bymeasuring the signal generated when the ligand binds to the cancer cellsin vivo.

5.8.1 Dosage and Frequency of Administration

The amount of an antibody, antibody conjugate or pharmaceuticalcomposition of the invention used in the prophylactic and/or therapeuticregimens which will be effective in the prevention, treatment, and/ormanagement of disorders characterized by cells expressing the IL-3receptor alpha subunit alpha subunit including cancer, can be determinedby methods disclosed herein. The frequency and dosage will vary alsoaccording to factors specific for each patient depending on the specificantibody or antibody conjugate administered, the severity of the(cancerous) condition, the route of administration, as well as age,body, weight, response, and the past medical history of the patient. Forexample, the dosage of an antibody or antibody conjugate of theinvention which will be effective in the treatment, prevention, and/ormanagement of cancer can be determined by administering the antibody orantibody conjugate to an animal model such as, e.g., the animal modelsdisclosed herein or known in to those skilled in the art. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges.

In some embodiments, the prophylactic and/or therapeutic regimenscomprise titrating the dosages administered to the patient so as toachieve a specified measure of therapeutic efficacy. Such measuresinclude a reduction in the amount of cancer stem cells in or from thepatient and/or a reduction in the amount of cancer cells in or from thepatient.

In some embodiments, the prophylactic and/or therapeutic regimenscomprise administering dosages and regimens of an antibody, antibodyconjugate or pharmaceutical composition of the invention that areeffective to reduce cancer stem cels. Methods that can be used todetermine the amount of cancer stem cells in a patient prior to, during,and/or following therapy are discussed infra in Section 5.9.

In certain embodiments, the dosage of the antibody, antibody conjugateof the invention in the prophylactic and/or therapeutic regimen isadjusted so as to achieve a reduction in the amount of cancer stem cellsfound in a test specimen extracted from a patient after undergoing thetherapeutic regimen, as compared with a reference sample. Here, thereference sample is a specimen extracted from the patient undergoingtherapy, wherein the specimen is extracted from the patient at anearlier time point. In one embodiment, the reference sample is aspecimen extracted from the same patient, prior to receiving theprophylactic or therapeutic regimen. In specific embodiments, the amountof cancer stem cells in the test specimen is at least 2%, 5%, 10%, 15%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% lower than in thereference sample.

In other embodiments, the dosage of the antibody or antibody conjugateof the invention in the prophylactic and/or therapeutic regimen isadjusted so as to achieve a reduction in the amount of cancer stem cellsfound in a test specimen extracted from a patient after undergoing theprophylactic and/or therapeutic regimen, as compared with a referencesample, wherein the reference sample specimen is extracted from ahealthy, noncancer-afflicted patient. In specific embodiments, theamount of cancer stem cells in the test specimen is at least within 60%,50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% of the amount of cancer stemcells in the reference sample.

In some embodiments, the dosage of the antibody or antibody conjugate ofthe invention in the prophylactic and/or therapeutic regimen is adjustedso as to achieve an amount of cancer stem cells that falls within apredetermined reference range. In these embodiments, the amount ofcancer stem cells in a test specimen is compared with a predeterminedreference range. In a specific embodiment, the predetermined referencerange is based on the amount of cancer stem cells obtained from apopulation(s) of patients suffering from the same type of cancer as thepatient undergoing the therapy.

In some embodiments, the prophylactic and/or therapeutic regimenscomprise administering dosages of the antibody, antibody conjugate orpharmaceutical composition of the invention that are effective to reducethe cancer cell population. Methods that can be used to determine thethe cancer cell population in a patient undergoing treatment arediscussed infra in Section 5.10.

In certain embodiments, the dosage of the antibody or antibody conjugateof the invention in the prophylactic and/or therapeutic regimen isadjusted so as to achieve a reduction in the amount of cancer cellsfound in a test specimen extracted from a patient after undergoing theprophylactic and/or therapeutic regimen, as compared with a referencesample. Here, the reference sample is a specimen extracted from thepatient undergoing therapy, wherein the specimen is extracted from thepatient at an earlier time point. In one embodiment, the referencesample is a specimen extracted from the same patient, prior to receivingthe prophylactic and/or therapeutic regimen. In specific embodiments,the amount of cancer cells in the test specimen is at least 2%, 5%, 10%,15%, 20%, 30%, 40%, 50%, or 60% lower than in the reference sample.

In some embodiments, the dosage of the antibody or antibody conjugate ofthe invention in the prophylactic and/or therapeutic regimen is adjustedso as to achieve an amount of cancer cells that falls within apredetermined reference range. In these embodiments, the amount ofcancer cells in a test specimen is compared with a predeterminedreference range.

In other embodiments, the dosage of the antibody or antibody conjugateof the invention in prophylactic and/or therapeutic regimen is adjustedso as to achieve a reduction in the amount of cancer cells found in atest specimen extracted from a patient after undergoing the prophylacticand/or therapeutic regimen, as compared with a reference sample, whereinthe reference sample is a specimen extracted from a healthy,noncancer-afflicted patient. In specific embodiments, the amount ofcancer cells in the test specimen is at least within 60%, 50%, 40%, 30%,20%, 15%, 10%, 5%, or 2% of the amount of cancer cells in the referencesample.

In treating certain human patients having solid tumors, extractingmultiple tissue specimens from a suspected tumor site may or may notprove impracticable. In these embodiments, the dosage of the compoundsof the invention in the prophylactic and/or therapeutic regimen for ahuman patient is extrapolated from doses in animal models that areeffective to reduce the amount of cancer stem cells in those animalmodels. In the animal models, the prophylactic and/or therapeuticregimens are adjusted so as to achieve a reduction in the amount ofcancer stem cells found in a test specimen extracted from an animalafter undergoing the prophylactic and/or therapeutic regimen, ascompared with a reference sample. The reference sample can be a specimenextracted from the same animal, prior to receiving the prophylacticand/or therapeutic regimen. In specific embodiments, the amount ofcancer stem cells in the test specimen is at least 2%, 5%, 10%, 15%,20%, 30%, 40%, 50%, or 60% lower than in the reference sample. The doseseffective in reducing the amount of cancer stem cells in the animals canbe normalized to body surface are (mg/m²) to provide an equivalent humandose.

The prophylactic and/or therapeutic regimens disclosed herein compriseadministration of an antibody or antibody conjugate of the invention orpharmaceutical compositions thereof to the patient in a single dose orin multiple doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or moredoses).

In one embodiment, the prophylactic and/or therapeutic regimens compriseadministration of an antibody or antibody conjugate of the invention orpharmaceutical compositions thereof in multiple doses. When administeredin multiple doses, the antibody, antibody conjugate or pharmaceuticalcompositions are administered with a frequency and in an amountsufficient to prevent, treat, and/or manage the condition. In oneembodiment, the frequency of administration ranges from once a day up toabout once every eight weeks. In another embodiment, the frequency ofadministration ranges from about once a week up to about once every sixweeks. In another embodiment, the frequency of administration rangesfrom about once every three weeks up to about once every four weeks. Incertain embodiments, the antibody or antibody conjugate is administeredover a period of one week to two years. In yet another embodiment, theantibody or antibody conjugate is administered over a period of twoweeks or greater. In other embodiments, the antibody or antibodyconjugate is administered over a period of two weeks to one year. Infurther embodiments, the antibody or antibody conjugate is administeredover a period of two weeks to six months. In some embodiments, theantibody or antibody conjugate is administered over a period of twoweeks to twelve weeks. In yet other embodiments, the antibody orantibody conjugate is administered over a period of two weeks to sixweeks. In certain embodiments, the antibody or antibody conjugate isadministered once a week, twice a week, three times a week, four times aweek, five times a week, six times a week, or seven times a week. Inpreferred embodiments, the antibody or antibody conjugate isadministered at least three times a week. In other preferredembodiments, the compound is administered daily for five consecutivedays, or daily for seven consecutive days. In other embodiments, theantibody or antibody conjugate is administered once a day, twice a day,three times a day, four times a day, or five times a day. In preferredembodiments, the antibody or antibody conjugate is administered threetimes a week over a period of two weeks. In some embodiments, each timethe antibody or antibody conjugate is administered, it is administeredat a dose of 4 μg/kg per day or greater. In some embodiments, thecompound is administered for one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen cycles.

In a specific embodiment, the antibody is administered at a dose of0.001 mg/kg per day to 100 mg/kg per day. In another embodiment, theantibody conjugate is administered at a dose of 0.001 mg/kg per day to10 mg/kg per day. In certain embodiments, the antibody is administeredat a dose of 0.1 mg/kg per day or greater. In other embodiments, theantibody is administered at a dose in a range of between about 0.01mg/kg per day to about 20 mg/kg per day.

In a specific embodiment, the conjugate is administered at a dose of0.001 mg/kg to 5 mg/kg per day. In specific embodiments, the conjugateis administered at a dose in a range of between about 4 μg/kg per day toabout 20 μg/kg per day. In further embodiments, the conjugate isadministered at doses up to and including the maximum tolerated dosewithout toxicity. In specific embodiments, where the disease is myeloidleukemia, the dosage given is in a range of between greater than 0.01mg/kg per day to about 60 mg/kg per day.

In some embodiments of the invention, the dosage of an antibody orantibody conjugate of the invention or pharmaceutical compositionthereof administered is at least 1.5, 1.6, 1.8, 2, 2.5, 3, 4, 5, 6, 7,8, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times lower than themaximum tolerated dose (MTD) over a period of three months, four months,six months, nine months, 1 year, 2 years, 3 years, 4 years or more.

In some embodiments of the invention, the dosage of an antibody orantibody conjugate of the invention or pharmaceutical compositionthereof administered is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2,2.5, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 timeslower than the human equivalent dose (HED) of the no observed adverseeffect level (NOAEL) over a period of three months, four months, sixmonths, nine months, 1 year, 2 years, 3 years, 4 years or more.

Generally, the dosage of an antibody of the invention administered to asubject to prevent, treat and/or manage cancer in the range of 0.001 to100 mg/kg, more typically, 0.1 to 20 mg/kg of the subject's body weight.

Generally, the dosage of a conjugate of the invention administered to asubject to prevent, treat, and/or manage cancer is in the range of0.0001 to 0.5 mg/kg, and more typically, in the range of 0.1 μg/kg to100 μg/kg, of the subject's body weight. In one embodiment, the dosageadministered to a subject is in the range of 0.1 μg/kg to 50 μg/kg, or 1μg/kg to 50 μg/kg, of the subject's body weight, more preferably in therange of 0.1 μg/kg to 25 μg/kg, or 1 μg/kg to 25 μg/kg, of the patient'sbody weight.

In a specific embodiment, the dosage of a conjugate of the inventionadministered to a subject to prevent, treat, and/or manage cancer in apatient is 500 μg/kg or less, preferably 250 μg/kg or less, 100 μg/kg orless, 95 μg/kg or less, 90 μg/kg or less, 85 μg/kg or less, 80 μg/kg orless, 75 μg/kg or less, 70 μg/kg or less, 65 μg/kg or less, 60 μg/kg orless, 55 μg/kg or less, 50 μg/kg or less, 45 μg/kg or less, 40 μg/kg orless, 35 μg/kg or less, 30 μg/kg or less, 25 μg/kg or less, 20 μg/kg orless, 15 μg/kg or less, 10 μg/kg or less, 5 μg/kg or less, 2.5 μg/kg orless, 2 μg/kg or less, 1.5 μg/kg or less, or 1 μg/kg or less of apatient's body weight. In another specific embodiment, the dosage of anantibody of the invention administered to a subject to prevent, treatand/or manage cancer in a patient is a unit dose of 2 mg, 5 mg, 10 mg,15 mg, 30 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500mg, 750 mg, 1000 mg, or 1 to 50 mg, 10 to 100 mg, 10 to 500 mg, 10 to1000 mg, 100 to 500 mg or 100 to 1000 mg.

In another specific embodiment, the dosage of a conjugate of theinvention administered to a subject to prevent, treat, and/or managecancer in a patient is a unit dose of 0.1 μg to 20 μg, 0.1 μg to 15 μg,0.1 μg to 12 μg, 0.1 μg to 10 μg, 0.1 μg to 8 μg, 0.1 μg to 7 μg, 0.1 μgto 5 μg, 0.1 to 2.5 μg, 0.25 μg to 20 μg, 0.25 to 15 μg, 0.25 to 12 μg,0.25 to 10 μg, 0.25 to 8 μg, 0.25 μg to 7 m g, 0.25 μg to 5 μg, 0.5 μgto 2.5 μg, 1 μg to 20 μg, 1 μg to 15 μg, 1 μg to 12 μg, 1 μg to 10 μg, 1μg to 8 μg, 1 μg to 7 μg, 1 μg to 5 μg, or 1 μg to 2.5 μg.

In a specific embodiment, the dosage of a conjugate of the inventionadministered to a subject to prevent, treat, and/or manage cancer in apatient is in the range of 0.01 to 10 g/m², and more typically, in therange of 0.1 g/m² to 7.5 g/m², of the subject's body weight. In oneembodiment, the dosage administered to a subject is in the range of 0.5g/m² to 5 g/m², or 1 g/m² to 5 g/m² of the subject's body's surfacearea.

In other embodiments, the prophylactic and/or therapeutic regimencomprises administering to a patient one or more doses of an effectiveamount of an antibody or antibody conjugate of the invention, whereinthe dose of an effective amount achieves a plasma level of at least 0.1μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least 15 μg/ml, atleast 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, atleast 200 μg/ml, at least 225 μg/ml, at least 250 μg/ml, at least 275μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, atleast 375 μg/ml, or at least 400 μg/ml of the antibody or antibodyconjugate of the invention.

In other embodiments, the prophylactic and/or therapeutic regimencomprises administering to a patient a plurality of doses of aneffective amount of an antibody or antibody conjugate of the invention,wherein the plurality of doses maintains a plasma level of at least 0.1μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least 15 μg/ml, atleast 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 1751 g/ml, atleast 200 μg/ml, at least 225 μg/ml, at least 250 μg/ml, at least 275μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, atleast 375 μg/ml, or at least 400 μg/ml of the antibody or antibodyconjugate of the invention for at least 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 15 months, 18 months, or 24 months.

In some embodiments, the prophylactic and/or therapeutic regimencomprises administration of an antibody or antibody conjugate of theinvention in combination with one or more additional anticancertherapeutics. In some embodiments, the dosages of the one or moreadditional anticancer therapeutics used in the combination therapy islower than those which have been or are currently being used to prevent,treat, and/or manage cancer. The recommended dosages of the one or moreadditional anticancer therapeutics currently used for the prevention,treatment, and/or management of cancer can be obtained from anyreference in the art including, but not limited to, Hardman et al.,eds., Goodman & Gilman's The Pharmacological Basis Of Basis OfTherapeutics, 10th ed., McGraw-Hill, New York, 2001; Physician's DeskReference (60th ed., 2006), which are incorporated herein by referencein its entirety.

5.8.2 Additional Therapies

Any therapy (e.g., therapeutic or prophylactic agent) which is useful,has been used, or is currently being used for the prevention, treatment,and/or management of a disorder characterized by IL3R alpha chainexpression (e.g., cancer) can be used in compositions and methods of theinvention. Therapies (e.g., therapeutic or prophylactic agents) include,but are not limited to, peptides, polypeptides, conjugates, nucleic acidmolecules, small molecules, mimetic agents, synthetic drugs, inorganicmolecules, and organic molecules. Non-limiting examples of cancertherapies include chemotherapies, radiation therapies, hormonaltherapies, and/or biological therapies/immunotherapies and surgery. Incertain embodiments, a prophylactically and/or therapeutically effectiveregimen of the invention comprises the administration of a combinationof therapies.

Examples of cancer therapies include, but not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene hydrochloride; bisnafide dimesylate; bisphosphonates (e.g.,pamidronate (Aredria), sodium clondronate (Bonefos), zoledronic acid(Zometa), alendronate (Fosamax), etidronate, ibandornate, cimadronate,risedromate, and tiludromate); bizelesin; bleomycin sulfate; brequinarsodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide;carbetimer; carboplatin; carmustine; carubicin hydrochloride;carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin;cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; EphA2 inhibitors; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine;gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride;ifosfamide; ilmofosine; interleukin II (including recombinantinterleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b;interferon alpha-n1; interferon alpha-n3; interferon beta-I a;interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotideacetate; letrozole; leuprolide acetate; liarozole hydrochloride;lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol;maytansine; mechlorethamine hydrochloride; anti-CD2 antibodies;megestrol acetate; melengestrol acetate; melphalan; menogaril;mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Other examples of cancer therapies include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; Bcl-2 inhibitors; Bcl-2 familyinhibitors, including ABT-737; BCR/ABL antagonists; benzochlorins;benzoylstaurosporine; beta lactam derivatives; beta-alethine;betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide;bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin;breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol;calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine;carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700;cartilage derived inhibitor; carzelesin; casein kinase inhibitors(ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenyl spiromustine;docetaxel; docosanol; dolasetron; doxifluridine; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; HMG CoA reductase inhibitors (e.g.,atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin,rosuvastatin, and simvastatin); hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; interferons; interleukins; iobenguane;iododoxorubicin; ipomeanol, 4-iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; LFA-3TIP (Biogen, Cambridge, Mass.; InternationalPublication No. WO 93/0686 and U.S. Pat. No. 6,162,432); liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RH retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;5-fluorouracil; leucovorin; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide;tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene bichloride; topsentin; toremifene; totipotent stem cellfactor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; thalidomide; velaresol; veramine; verdins; verteporfin;vinorelbine; vinxaltine; vorozole; zanoterone; zeniplatin; zilascorb;and zinostatin stimalamer.

In some embodiments, the therapy(ies) used in combination with anantibody or antibody conjugate of the invention is an immunomodulatoryagent. Non-limiting examples of immunomodulatory agents includeproteinaceous agents such as cytokines, peptide mimetics, and antibodies(e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs,Fab or F(ab)2 fragments or epitope binding fragments), nucleic acidmolecules (e.g., antisense nucleic acid molecules and triple helices),small molecules, organic compounds, and inorganic compounds. Inparticular, immunomodulatory agents include, but are not limited to,methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran,cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506(tacrolimus)), methylprednisolone (MP), corticosteroids, steroids,mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide).Other examples of immunomodulatory agents can be found, e.g., in U.S.Publ'n No. 2005/0002934 A1 at paragraphs 259-275 which is incorporatedherein by reference in its entirety. In one embodiment, theimmunomodulatory agent is a chemotherapeutic agent. In an alternativeembodiment, the immunomodulatory agent is an immunomodulatory agentother than a chemotherapeutic agent. In some embodiments, thetherapy(ies) used in accordance with the invention is not animmunomodulatory agent.

In some embodiments, the therapy(ies) used in combination with anantibody or antibody conjugate of the invention is an anti-angiogenicagent. Non-limiting examples of anti-angiogenic agents include proteins,polypeptides, peptides, conjugates, antibodies (e.g., human, humanized,chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab)2fragments, and antigen-binding fragments thereof) such as antibodiesthat bind to TNF-alpha, nucleic acid molecules (e.g., antisensemolecules or triple helices), organic molecules, inorganic molecules,and small molecules that reduce or inhibit angiogenesis. Other examplesof anti-angiogenic agents can be found, e.g., in U.S. Publ′n No.2005/0002934 A1 at paragraphs 277-282, which is incorporated byreference in its entirety. In other embodiments, the therapy(ies) usedin accordance with the invention is not an anti-angiogenic agent.

In some embodiments, the therapy(ies) used in combination with anantibody or antibody conjugate of the invention is an inflammatoryagent. Non-limiting examples of anti-inflammatory agents include anyanti-inflammatory agent, including agents useful in therapies forinflammatory disorders, well-known to one of skill in the art.Non-limiting examples of anti-inflammatory agents include non-steroidalanti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs,anticholinergics (e.g., atropine sulfate, atropine methylnitrate, andipratropium bromide (ATROVENT™)), beta2-agonists (e.g., abuterol(VENTOLIN™ and PROVENTIL™), bitolterol (TORNALATE™), levalbuterol(XOPONEX™), metaproterenol (ALUPENT™), pirbuterol (MAXAIR™), terbutlaine(BRETHAIRE™ and BRETHINE™), albuterol (PROVENTIL™, REPETABS™, andVOLMAX™), formoterol (FORADIL AEROLIZER™), and salmeterol (SEREVENT™ andSEREVENT DISKUS™)), and methylxanthines (e.g., theophylline (UNIPHYL™,THEO-DUR™, SLO-BID™, AND TEHO-42™)). Examples of NSAIDs include, but arenot limited to, aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac(VOLTAREN™), etodolac (LODINE™) fenoprofen (NALFON™), indomethacin(INDOCIN™), ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone(RELAFEN™), sulindac (CLINORIL™) tolmentin (TOLECTIN™), rofecoxib(VIOXX™), naproxen (ALEVE™ NAPROSYN™), ketoprofen (ACTRON™) andnabumetone (RELAFEN™). Such NSAIDs function by inhibiting acyclooxygenase enzyme (e.g., COX-1 and/or COX-2). Examples of steroidalanti-inflammatory drugs include, but are not limited to,glucocorticoids, dexamethasone (DECADRON™), corticosteroids (e.g.,methylprednisolone (MEDROL™)), cortisone, hydrocortisone, prednisone(PREDNISONE™ and DELTASONE™), prednisolone (PRELONE™ and PEDIAPRED™),triamcinolone, azulfidine, and inhibitors of eicosanoids (e.g.,prostaglandins, thromboxanes, and leukotrienes. Other examples ofanti-inflammatory agents can be found, e.g., in U.S. Publ′n No.005/0002934 A1 at paragraphs 290-294, which is incorporated by referencein its entirety. In other embodiments, the therapy(ies) used inaccordance with the invention is not an anti-inflammatory agent.

In certain embodiments, the therapy(ies) used is an alkylating agent, anitrosourea, an antimetabolite, and anthracyclin, a topoisomerase IIinhibitor, or a mitotic inhibitor. Alkylating agents include, but arenot limited to, busulfan, cisplatin, carboplatin, chlorambucil,cyclophosphamide, ifosfamide, decarbazine, mechlorethamine, melphalan,and themozolomide. Nitrosoureas include, but are not limited tocarmustine (BCNU) and lomustine (CCNU). Antimetabolites include but arenot limited to 5-fluorouracil, capecitabine, methotrexate, gemcitabine,cytarabine, and fludarabine. Anthracyclines include but are not limitedto daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone.Topoisomerase II inhibitors include, but are not limited to, topotecan,irinotecan, etoposide (VP-16), and teniposide. Mitotic inhibitorsinclude, but are not limited to taxanes (paclitaxel, docetaxel), and thevinca alkaloids (vinblastine, vincristine, and vinorelbine).

The invention includes the use of agents that target cancer stem cellsin combination with an antibody or antibody conjugate of the invention.In some embodiments, the agent used is an agent that binds to a marker,e.g., antigen on cancer stem cells. In a specific embodiment, the agentbinds to an antigen that is expressed at a greater level on cancer stemcells than on normal stem cells. In a specific embodiment, the agentbinds specifically to a cancer stem cell antigen. In other embodiments,the therapy(ies) used in accordance with the invention is an agent thatbinds to a marker on cancer stem cells. Non-limiting examples ofantigens on cancer stem cells that can be used to target cancer stemcells include CD34+, CD44+, CD133+, CD34+, CD19+, CD20+, CD47+, CD96+,CD133+, and α2β1hi. In one embodiment, the agent that binds to a markeron cancer stem cells is an antibody. In another embodiment, the agentthat binds to a marker on cancer stem cells is a ligand. In certainembodiments, the antibody or ligand is attached directly or indirectlyto a therapeutic moiety. Non-limiting examples of therapeutic moietiesinclude, but are not limited to, therapeutic enzymes, chemotherapeuticagents, cytokines, radionuclides, antimetabolites, and toxins.

In certain embodiments, antibodies that bind to a marker on cancer stemcells are substantially non-immunogenic in the treated subject.Non-immunogenic antibodies include, but are not limited to, making theantibody chimeric, humanizing the antibody, and antibodies from the samespecies as subject receiving the therapy. Antibodies that bind tomarkers in cancer stem cells can be produced using techniques known inthe art. See, for example, paragraphs 539-573 of U.S. Publ'n No.2005/0002934 A1, which is incorporated by reference in its entirety.

In some embodiments, an antibody or antibody conjugate of the inventionis used in combination with radiation therapy comprising the use ofx-rays, gamma rays and other sources of radiation to destroy cancer stemcells and/or cancer cells. In specific embodiments, the radiationtherapy is administered as external beam radiation or teletherapy,wherein the radiation is directed from a remote source. In otherembodiments, the radiation therapy is administered as internal therapyor brachytherapy wherein a radioactive source is placed inside the bodyclose to cancer stem cells, cancer cells and/or a tumor mass.

Currently available cancer therapies and their dosages, routes ofadministration and recommended usage are known in the art and have beendescribed in such literature as the Physician's Desk Reference (60thed., 2006). In accordance with the present invention, the dosages andfrequency of administration of chemotherapeutic agents are describedsupra.

5.8.3 Types of Disease and Disorders

The present invention provides methods of treating or preventing ormanaging a disease or disorder characterized by cells expressing theIL-3 receptor alpha subunit in humans by administering to humans in needof such treatment or prevention a pharmaceutical composition comprisingan amount of antibody or an antibody conjugate of the inventioneffective to treat or prevent the disease or disorder. In certainembodiments, the disease or disorder is not a hematologic cancer. Inother embodiments, the disease or disorder is an allergic disease ordisorder. In other embodiments, the disease or disorder is aninflammatory disease or disorder. In another embodiment, the disease ordisorder is one characterized as affecting plasmacytoid dendritic cells(e.g., dentritic cell cancers such as NK blastic leukemia and CD4⁺CD56⁺dermatologic neoplasm). In certain embodiments, the subjects have acutemyelogenous leukemia (AML). In certain other embodiments, the subjectshave myelodysplastic syndrome (MDS). In other embodiments, the subjectshave chronic myelomonocytic leukemia (CMML), CML, ALL, hairy cellleukemia, Hodgkin's disease, or non-Hodgkin's lymphoma.

In certain embodiments, the present invention encompasses treatingpatients with NK blastic leukemia and/or lymphoma (also known asplasmacytoid dendritic cell lymphoma) by administering to those patientsan antibody or an antibody conjugate of the invention after thosepatients have gone into remission following another cancer therapy. Incertain embodiments, for those patients in remission, an effectiveamount of the antibody or antibody conjugate will be an amount effectiveto prolong or increase the amount of time before recurrence of thecancer.

The present invention encompasses methods for preventing, treating,managing, and/or ameliorating an inflammatory disorder or one or moresymptoms thereof as an alternative to other conventional therapies. Inspecific embodiments, the patient being managed or treated in accordancewith the methods of the invention is refractory to other therapies or issusceptible to adverse reactions from such therapies. The patient may bea person with a suppressed immune system (e.g., post-operative patients,chemotherapy patients, and patients with immunodeficiency disease,patients with broncho-pulmonary dysplasia, patients with congenitalheart disease, patients with cystic fibrosis, patients with acquired orcongenital heart disease, and patients suffering from an infection), aperson with impaired renal or liver function, the elderly, children,infants, infants born prematurely, persons with neuropsychiatricdisorders or those who take psychotropic drugs, persons with historiesof seizures, or persons on medication that would negatively interactwith conventional agents used to prevent, manage, treat, or ameliorate aviral respiratory infection or one or more symptoms thereof.

In an embodiment of the invention, diseases that are characterized byplasmacytoid dendritic cells, which cells demonstrate high expression ofthe alpha chain of the IL-3 receptor, are targeted. Such diseasesinclude, but are not limited to, HIV, herpes, CMV, autoimmune diseases,and cancers including but not limited to NK blastic lymphoma, dendriticcell cancer including plasmacytoid dendritic cell cancer, anddermatologic neoplasms.

Autoimmune Disorders

In certain embodiments, the invention provides a method of preventing,treating, managing, and/or ameliorating an autoimmune disorder or one ormore symptoms thereof, said method comprising administering to a subjectin need thereof a dose of an effective amount of one or morepharmaceutical compositions of the invention, wherein the cells involvedin such disorders express the interleukin-3 receptor beta subunit. Inautoimmune disorders, the immune system triggers an immune response andthe body's normally protective immune system causes damage to its owntissues by mistakenly attacking self. There are many differentautoimmune disorders which affect the body in different ways. Forexample, the brain is affected in individuals with multiple sclerosis,the gut is affected in individuals with Crohn's disease, and thesynovium, bone and cartilage of various joints are affected inindividuals with rheumatoid arthritis. As autoimmune disorders progress,destruction of one or more types of body tissues, abnormal growth of anorgan, or changes in organ function may result. The autoimmune disordermay affect only one organ or tissue type or may affect multiple organsand tissues. Organs and tissues commonly affected by autoimmunedisorders include red blood cells, blood vessels, connective tissues,endocrine glands (e.g., the thyroid or pancreas), muscles, joints, andskin.

Examples of autoimmune disorders that can be prevented, treated,managed, and/or ameliorated by the methods of the invention include, butare not limited to, adrenergic drug resistance, alopecia areata,ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison'sdisease, autoimmune diseases of the adrenal gland, allergicencephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune inflammatory eye disease, autoimmune neonatalthrombocytopenia, autoimmune neutropenia, autoimmune oophoritis andorchitis, autoimmune thrombocytopenia, autoimmune thyroiditis, Behcet'sdisease, bullous pemphigoid, cardiomyopathy, cardiotomy syndrome, celiacsprue-dermatitis, chronic active hepatitis, chronic fatigue immunedysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, dense depositdisease, discoid lupus, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, glomerulonephritis (e.g., IgA nephrophathy),gluten-sensitive enteropathy, Goodpasture's syndrome, Graves' disease,Guillain-Barre, hyperthyroidism (i.e., Hashimoto's thyroiditis),idiopathic pulmonary fibrosis, idiopathic Addison's disease, idiopathicthrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis,lichen planus, lupus erythematosus, Meniere's disease, mixed connectivetissue disease, multiple sclerosis, Myasthenia Gravis, myocarditis, type1 or immune-mediated diabetes mellitus, neuritis, other endocrine glandfailure, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, Polyendocrinopathies, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, post-MI,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynauld's phenomenon, relapsing polychondritis,Reiter's syndrome, rheumatic heart disease, rheumatoid arthritis,sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome,systemic lupus erythematosus, takayasu arteritis, temporalarteritis/giant cell arteritis, ulcerative colitis, urticaria, uveitis,Uveitis Opthalmia, vasculitides such as dermatitis herpetiformisvasculitis, vitiligo, and Wegener's granulomatosis.

Allergies

In certain embodiments, the invention provides a method of preventing,treating, managing, and/or ameliorating one or more allergic diseases orallergies or one or more symptoms thereof, wherein the cells involved insuch diseases or allergies express the interleukin-3 receptor betasubunit, said method comprising administering to a subject in needthereof a dose of an effective amount of one or more pharmaceuticalcompositions of the invention. Immune-mediated allergic(hypersensitivity) reactions are classified into four types (I-IV)according to the underlying mechanisms leading to the manifestation ofthe allergic symptoms. Type I allergic reactions are immediatehypersensitivity reactions characterized by IgE-mediated release ofvasoactive substances such as histamine from mast cells and basophils.Over hours, the mast cells and basophils release proinflammatorycytokines producing vasodilation, increased capillary permeability,glandular hypersecretion, smooth muscle spasm, and tissue infiltrationwith eosinophils and other inflammatory cells.

Type II allergic reactions are cytotoxic hypersensitivity reactions andinvolve IgG or IgM antibodies bound to cell surface antigens withsubsequent complement fixation. Certain cytotoxic cells, such as killerT cells or macrophages, are activated, bind to cells coated with IgG anddestroy the target cells. Type II reactions may result in cytolysis ortissue damage.

Type III reactions are immune-complex reactions resulting from depositsof circulating antigen-antibody immune complexes in blood vessels ortissues. Acute inflammation results from the immune-complex initiating asequence of events that results in polymorphonuclear cell migration andrelease of lysosomal proteolytic enzymes and permeability factors intissues.

Type IV reactions are delayed hypersensitivity reactions caused bysensitized T lymphocytes after contact with a specific antigen.Activated sensitized T lymphocytes cause immunologic injury by directtoxic effect or through release of lymphokines and other solublesubstances. The activated T lymphocytes may also release cytokines thataffect the activity of macrophages, neutrophils, and lymphoid killercells.

Allergic reactions can be immediate, late-phase, or chronic. Continuousor chronic exposure to an allergen can result in chronic allergicinflammation. Tissues of sites of chronic inflammation containeosinophils and T cells that release mediators that can cause tissuedamage, increased inflammation, and increased sensitivity.

Currently, allergic reactions are treated with drugs such asantihistamines, corticosteroids, vasodilators, bronchodilators,leukotriene inhibitors, and immunomodulators which attempt to alleviatethe symptoms associated with the allergic reaction.

Cancer

Any type of cancer in which the cancer stem cells or cancer cellsexpress the interleukin-3 receptor alpha subunit can be prevented,treated, and/or managed in accordance with the invention. Non-limitingexamples of cancers that can be prevented, treated, and/or managed inaccordance with the invention include: leukemias, such as but notlimited to, acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemias, such as, myeloblastic, promyelocytic, myelomonocytic,monocytic, and erythroleukemia leukemias and myelodysplastic syndrome;chronic leukemias, such as but not limited to, chronic myelocytic(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cellleukemia; polycythemia vera; lymphomas such as but not limited toHodgkin's disease, non-Hodgkin's disease; multiple myelomas such as butnot limited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; dendritic cell cancer, including plasmacytoiddendritic cell cancer, NK blastic lymphoma (also known as cutaneousNK/T-cell lymphoma and agranular (CD4+/CD56+) dermatologic neoplasms);basophilic leukemia; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors such as but not limited to, glioma, astrocytoma, brain stemglioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to ductal carcinoma, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer such as but notlimited to pheochromocytom and adrenocortical carcinoma; thyroid cancersuch as but not limited to papillary or follicular thyroid cancer,medullary thyroid cancer and anaplastic thyroid cancer; pancreaticcancer such as but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor; esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cysticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungating (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma; gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to papillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, prostaticintraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers such as but notlimited to renal cell carcinoma, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

The prophylactically and/or therapeutically effective regimens are alsouseful in the treatment, prevention and/or management of a variety ofcancers or other abnormal proliferative diseases wherein the cells ofsuch diseases express the interleukin-3 receptor beta subunit, including(but not limited to) the following: carcinoma, including that of thebladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach,cervix, thyroid and skin; including squamous cell carcinoma;hematopoietic tumors of lymphoid lineage, including leukemia, acutelymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, Tcell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloidlineage, including acute and chronic myelogenous leukemias andpromyelocytic leukemia; tumors of mesenchymal origin, includingfibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma,seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of thecentral and peripheral nervous system, including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin,including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and othertumors, including melanoma, xeroderma pigmentosum, keratoactanthoma,seminoma, thyroid follicular cancer and teratocarcinoma. In someembodiments, cancers associated with aberrations in apoptosis areprevented, treated and/or managed in accordance with the methods of theinvention. Such cancers may include, but not be limited to, follicularlymphomas, carcinomas with p53 mutations, hormone dependent tumors ofthe breast, prostate and ovary, and precancerous lesions such asfamilial adenomatous polyposis, and myelodysplastic syndromes. Inspecific embodiments, malignancy or dysproliferative changes (such asmetaplasias and dysplasias), or hyperproliferative disorders of theskin, lung, liver, bone, brain, stomach, colon, breast, prostate,bladder, kidney, pancreas, ovary, and/or uterus are prevented, treatedand/or managed in accordance with the methods of the invention. In otherspecific embodiments, a sarcoma, melanoma, or leukemia is prevented,treated and/or managed in accordance with the methods of the invention.In certain embodiments, the subjects have acute myelogenous leukemia(AML). In certain other embodiments, the subjects have myelodysplasticsyndrome (MDS). In other embodiments, the subjects have chronicmyelomonocytic leukemia (CMML). In other specific embodiments,myelodysplastic syndrome is prevented, treated and/or managed inaccordance with the methods of the invention.

5.8.4 Target Patient Populations

In accordance with the invention, the pharmaceutical compositions of thepresent invention are administered to humans in need of inhibition ofcells that express the alpha subunit of the interleukin-3 receptor. Incertain embodiments, the growth of such cells is inhibited. In otherembodiments, the conjugates of the present invention are administered tohumans with diseases and disorders associated with overexpression of theIL-3 receptor. In certain embodiments, the subject does have myeloidleukemia. In other embodiments, the disease or disorder is an allergicdisease or disorder. In some embodiments, the disease or disorder is anautoimmune disease. In certain embodiments, the subjects have acutemyelogenous leukemia (AML). In certain other embodiments, the subjectshave myelodysplastic syndrome (MDS). In other embodiments, the subjectshave chronic myelomonocytic leukemia (CMML).

In accordance with the invention, pharmaceutical compositions of thepresent invention are administered to subjects developing, developed, orexpected to develop cancer (e.g., subjects with a genetic predispositionfor a particular type of cancer, subjects that have been exposed to acarcinogen, subjects with newly diagnosed cancer, subjects that havefailed treatment for cancer, subjects who have relapsed from cancer, orsubjects that are in remission from a particular cancer). Such subjectsmay or may not have been previously treated for cancer or may be inremission, relapsed, or may have failed treatment. Such patients mayalso have abnormal cytogenetics. The pharmaceutical compositions may beused as any line of cancer therapy, e.g., a first line, second line, orthird line of cancer therapy. In a specific embodiment, the subject toreceive or receiving a pharmaceutical composition of the invention isreceiving or has received other cancer therapies. In another embodimentthe subject to receive a pharmaceutical composition of the invention isreceiving other cancer therapies and pharmaceutical compositions of theinvention are administered to the subject before any adverse effects orintolerance of these other cancer therapies occurs. In an alternativeembodiment, the subject to receive or receiving a pharmaceuticalcomposition of the invention has not received or is not receiving othercancer therapies.

In a specific embodiment, the subject has been diagnosed with cancerusing techniques known to one of skill in the art including, but notlimited to, physical examination (e.g., prostate examination, breastexamination, lymph nodes examination, abdominal examination, skinsurveillance, general palpation), visual methods (e.g., colonoscopy,bronchoscopy, endoscopy), PAP smear analyses (cervical cancer), stoolguaiac analyses, blood tests (e.g., complete blood count (CBC) test,prostate specific antigen (PSA) test, carcinoembryonic antigen (CEA)test, cancer antigen (CA)-125 test, alpha-fetoprotein (AFP), liverfunction tests), karyotyping analyses, bone marrow analyses (e.g., incases of hematological malignancies), histology, flow cytometry,cytology, a sputum analysis and imaging methods (e.g., computedtomography (CT), magnetic resonance imaging (MRI), ultrasound, X-rayimaging, mammography, PET scans, radionuclide scans, bone scans).Subjects may or may not have been previously treated for cancer.

In one embodiment, a pharmaceutical composition of the invention isadministered to a subject that is undergoing or has undergone surgery toremove a tumor neoplasm. In a specific embodiment, a pharmaceuticalcomposition of the invention is administered to a subject concurrentlyor following surgery to remove a tumor or neoplasm. In anotherembodiment, a pharmaceutical composition of the invention isadministered to a subject before surgery to remove a tumor or neoplasmand, in some embodiments, during and/or after surgery.

In one embodiment, a pharmaceutical composition of the invention isadministered to a subject after a course of therapy with the goal ofkilling cancer cells. In some embodiments, the course of therapyinvolves the administration of bolus doses of chemotherapeutic agentsand/or bolus doses of radiation therapy. In a specific embodiment, apharmaceutical composition of the invention is administered to a subjectafter the subject has received a course of therapy involving a dosewhich is at, or is below, the maximum tolerated dose or the no observedadverse effect level doses of one or more chemotherapeutic agents and/orradiation therapy.

In certain embodiments, a pharmaceutical composition of the invention isadministered to a subject as an alternative to chemotherapy, radiationtherapy, hormonal therapy, surgery, small molecule therapy,anti-angiogenic therapy, differentiation therapy, epigenetic therapy,radioimmunotherapy, targeted therapy, and/or biological therapyincluding immunotherapy where the therapy has proven or may prove tootoxic, i.e., results in unacceptable or unbearable side effects for thesubject. In some embodiments, a prophylactically and/or therapeuticallyeffective regimen is administered to a subject that is susceptible toadverse reactions from other cancer therapies. The subject may, e.g.,have a suppressed immune system (e.g., post-operative patients,chemotherapy patients, and patients with immunodeficiency disease), havean impaired renal or liver function, be elderly, be a child, be aninfant, have a neuropsychiatric disorder, take a psychotropic drug, havea history of seizures, or be on medication that would negativelyinteract with the cancer therapies.

In a specific embodiment, a pharmaceutical composition of the inventionis administered to subjects that will, are or have radiation therapy.Among these subjects are those that have received chemotherapy, hormonaltherapy, small molecule therapy, anti-angiogenic therapy,differentiation therapy, targeted therapy, radioimmunotherapy,epigenetic therapy, and/or biological therapy, including immunotherapyas well as those who have undergone surgery.

In another embodiment, a pharmaceutical composition of the invention isadministered to subjects that will, are, or have received hormonaltherapy and/or biological therapy, including immunotherapy. Among thesesubjects are those that have received chemotherapy, small moleculetherapy, anti-angiogenic therapy, differentiation therapy, targetedtherapy, radioimmunotherapy, epigenetic therapy, and/or radiationtherapy as well as those who have undergone surgery.

In certain embodiments, a pharmaceutical composition of the invention isadministered to a subject refractory to one or more therapies. In oneembodiment, that a cancer is refractory to a therapy means that at leastsome significant portion of the cancer cells are not killed or theircell division is not arrested. The determination of whether the cancercells are refractory can be made either in vivo or in vitro by anymethod known in the art for assaying the effectiveness of a therapy oncancer cells, using the art-accepted meanings of “refractory” in such acontext. In various embodiments, a cancer is refractory where the amountof cancer cells has not been significantly reduced, or has increased. Inother embodiments, that a cancer is refractory means that at least somesignificant portion of cancer stem cells are not killed or their celldivision arrested. The determination of whether the cancer stem cellsare refractory can be made either in vivo or in vitro by any methodsknown in the art or described herein.

In some embodiments, a pharmaceutical composition of the invention isadministered to reverse the resistance to, or increase the sensitivityof cancer cells to certain hormonal, radiation and chemotherapeuticagents thereby resensitizing the cancer cells to one or more of theseagents, which can then be administered (or continue to be administered)to treat or manage cancer, including to prevent metastasis. In aspecific embodiment, the regimens of the invention are administered topatients with increased levels of the cytokine IL-6, which has beenassociated with the development of cancer cell resistance to differenttreatment regimens, such as chemotherapy and hormonal therapy.

In some embodiments, a pharmaceutical composition of the invention isadministered to a subject with a mean absolute lymphocyte count of atleast approximately 400 cells/mm³, at least 500 cells/mm³, at leastapproximately 600 cells/mm³, at least approximately 700 cells/mm³, atleast approximately 800 cells/mm³, at least approximately 900 cells/mm³,at least approximately 1000 cells/mm³, at least approximately 1100cells/mm³, at least approximately 1200 cells/mm³. In other embodiments,a prophylactically and/or therapeutically effective regimen of theinvention is administered to a subject with a mean absolute lymphocytecount of approximately 400 cells/mm³ to approximately 1200 cells/mm³,approximately 500 cells/mm³ to approximately 1200 cells/mm³,approximately 600 cells/mm³ to approximately 1200 cells/mm³,approximately 700 cells/mm³ to approximately 1200 cells/mm³,approximately 800 cells/mm³ to approximately 1200 cells/mm³,approximately 900 cells/mm³ to approximately 1200 cells/mm³,approximately 1000 cells/mm³ to approximately 1200 cells/mm³. In a morespecific embodiment, the regimen results in a mean absolute lymphocytecount of at least approximately 400 cells/mm³.

In some embodiments, a pharmaceutical composition of the invention isadministered to a subject that is in remission. In a specificembodiment, the subject has no detectable cancer, i.e., no cancer isdetectable using a conventional method described herein (e.g., MRI) orknown to one of skill in the art. In another embodiment, apharmaceutical composition of the invention is administered to a patientthat does not have a detectable immune response to diphtheria toxin. Ina preferred embodiment, the immune response is detected by ELISA.

5.8.5 Combination Therapies

The present invention also provides methods for preventing, treating,and/or managing cancer, the methods comprising administering to apatient (e.g., a human patient) in need thereof, a prophylacticallyand/or a therapeutically effective regimen, the regimen comprisingadministering to the patient a pharmaceutical composition of theinvention and one or more additional therapies, said additional therapynot being an antibody or antibody conjugate of the invention. In aspecific embodiment, the combination therapies of the invention comprisea pharmaceutical composition in accordance with the invention and atleast one other therapy that has the same mechanism of action as saidantibody or antibody conjugate. In another specific embodiment, thecombination therapies of the invention comprise a pharmaceuticalcomposition identified in accordance with the methods of the inventionand at least one other therapy (e.g., prophylactic or therapeutic agent)which has a different mechanism of action than said antibody or antibodyconjugate. The pharmaceutical composition of the invention and theadditional therapy can be administered separately, concurrently, orsequentially. The combination of agents can act additively orsynergistically. The combination therapies of the present inventionreduce the side effects associated with the therapies (e.g.,prophylactic or therapeutic agents).

The prophylactic or therapeutic agents of the combination therapies canbe administered to a subject in the same pharmaceutical composition.Alternatively, the prophylactic or therapeutic agents of the combinationtherapies can be administered concurrently to a subject in separatepharmaceutical compositions. The prophylactic or therapeutic agents maybe administered to a subject by the same or different routes ofadministration.

Any therapy (e.g., therapeutic or prophylactic agent) which is useful,has been used, or is currently being used for the prevention, treatment,and/or management of cancer can be used in compositions and methods ofthe invention. Therapies (e.g., therapeutic or prophylactic agents)include, but are not limited to, peptides, polypeptides, antibodies,conjugates, nucleic acid molecules, small molecules, mimetic agents,synthetic drugs, inorganic molecules, and organic molecules.Non-limiting examples of cancer therapies include chemotherapy,radiation therapy, hormonal therapy, surgery, small molecule therapy,anti-angiogenic therapy, differentiation therapy, epigenetic therapy,radioimmunotherapy, targeted therapy, and/or biological therapyincluding immunotherapy. In certain embodiments, a prophylacticallyand/or therapeutically effective regimen of the invention comprises theadministration of a combination of therapies.

Examples of cancer therapies include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthracyclin; anthramycin; asparaginase;asperlin; azacitidine (Vidaza); azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bisphosphonates (e.g., pamidronate (Aredria), sodium clondronate(Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate,ibandornate, cimadronate, risedromate, and tiludromate); bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine (Ara-C); dacarbazine; dactinomycin;daunorubicin hydrochloride; decitabine (Dacogen); demethylation agents,dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; EphA2 inhibitors; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine;histone deacetylase inhibitors (HDACs) gemcitabine hydrochloride;hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; imatinibmesylate (Gleevec, Glivec); interleukin II (including recombinantinterleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b;interferon alpha-n1; interferon alpha-n3; interferon beta-I a;interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotideacetate; lenalidomide (Revlimid); letrozole; leuprolide acetate;liarozole hydrochloride; lometrexol sodium; lomustine; losoxantronehydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride;anti-CD2 antibodies (e.g., siplizumab (MedImmune Inc.; InternationalPublication No. WO 02/098370, which is incorporated herein by referencein its entirety)); megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxaliplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Other examples of cancer therapies include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenyl spiromustine;docetaxel; docosanol; dolasetron; doxifluridine; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; HMG CoA reductase inhibitors (e.g.,atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin,rosuvastatin, and simvastatin); hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; interferons; interleukins; iobenguane;iododoxorubicin; ipomeanol, 4-iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; LFA-3TIP (Biogen, Cambridge, Mass.; InternationalPublication No. WO 93/0686 and U.S. Pat. No. 6,162,432); liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;5-fluorouracil; leucovorin; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide;tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene bichloride; topsentin; toremifene; totipotent stem cellfactor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; thalidomide; velaresol; veramine; verdins; verteporfin;vinorelbine; vinxaltine; VITAXIN™ (see U.S. Patent Pub. No. US2002/0168360 A1, dated Nov. 14, 2002, entitled “Methods of Preventing orTreating Inflammatory or Autoimmune Disorders by Administering Integrinαvβ3 Antagonists in Combination With Other Prophylactic or TherapeuticAgents”); vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

A non-limiting list of compounds that could be used to target cancerstem cells includes: inhibitors of interleukin-3 receptor (IL-3R) andCD123 (including peptides, peptide-conjugates, antibodies,antibody-conjugates, antibody fragments, and antibodyfragment-conjugates that target IL-3R or CD123); cantharidin;norcantharidin and analogs and derivatives thereof; Notch pathwayinhibitors including gamma secretase inhibitors; sonichedgehog/smoothened pathway inhibitors including cyclopamine and analogsthereof; antibodies to CD96; certain NF-kB/proteasome inhibitorsincluding parthenolide and analogs thereof; certain triterpenesincluding celastrol; certain mTOR inhibitors; compounds and antibodiesthat target the urokinase receptor; sinefungin; certain inosinemonophosphate dehydrogenase (IMPDH) inhibitors; PPAR-alpha andPPAR-gamma agonists and antagonists (including pioglitazone,tesaslitazar, muraglitazar, peliglitazar, lobeglitazone, balaglitazone,ragaglitazar, rosiglitazone, farglitazar, sodelglitazar, reglitazar,naveglitazar, oxeglitazar, metaglidasen, netoglitazone, darglitazone,englitazone, thiazolidinediones, aleglitazar, edaglitazone,rivoglitazone, troglitazone, imiglitazar, and sipoglitazar); telomeraseinhibitors; antibodies to EpCAM (ESA); GSK-3 beta agonists andantagonists (including Lithium, 6-bromoinirubin-3′-oxime (BIO), TDZD8);Wnt pathway inhibitors including antibodies to frizzled or smallmolecules that inhibit disheveled/frizzled or beta catenin; anti-CD20antibodies and conjugates (e.g. Rittman, Bexxar, Zevalin) for novel usein multiple myeloma or melanoma; anti-CD133 antibody; anti-CD44antibody; antibodies to IL-4; certain differentiation agents such asversnarinone; compounds that target CD33 such as an antibody orbetulinic acid; compounds that target lactadherin such as an antibody;small molecules or antibodies that target CXCR4 or SDF-1; smallmolecules or antibodies that target multi-drug resistance pumps;inhibitors of survivin; inhibitors of XIAP; small molecules that targetBcl-2; antibodies to CLL-1; and furin inhibitors (such ascucurbitacins).

An additional non-limiting list of compounds that could also be used totarget cancer stem cells includes i) antibodies, antibody fragments, andproteins that are either naked or conjugated to a therapeutic moietythat target certain cell surface targets on cancer stem cells, or ii)small molecules known in the art including ones that can be furtheroptimized (e.g. via chemistry) or identified via a cancer stemcell-based screen (e.g. such as one that would determine whether acompound impairs proliferation or viability of a cancer stem cellthrough standard methods, the cell surface and intracellular targetsincluding (not meant to be exhaustive) are: Rex1 (Zfp42), CTGF, ActivinA, Wnt, FGF-2, HIF-1, AP-2gamma, Bmi-1, nucleostemin, hiwi, Moz-TIF2,Nanog, beta-arrestin-2, Oct-4, Sox2, stella, GDF3, RUNX3, EBAF, TDGF-1,nodal, ZFPY, PTNE, Evi-1, Pax3, Mcl-1, c-kit, Lex-1, Zfx, lactadherin,aldehyde dehydrogenase, BCRP, telomerase, CD133, Bcl-2, CD26, Gremlin,and FoxC2.

In some embodiments, the therapy(ies) used in combination with acompound of the invention is an immunomodulatory agent. Non-limitingexamples of immunomodulatory agents include proteinaceous agents such ascytokines, peptide mimetics, and antibodies (e.g., human, humanized,chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments orepitope binding fragments), nucleic acid molecules (e.g., antisensenucleic acid molecules and triple helices), small molecules, organiccompounds, and inorganic compounds. In particular, immunomodulatoryagents include, but are not limited to, methotrexate, leflunomide,cyclophosphamide, cytoxan, Immuran, cyclosporine A, minocycline,azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone(MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin(sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamindes (e.g., leflunamide), T cell receptor modulators,cytokine receptor modulators, and modulators mast cell modulators. Otherexamples of immunomodulatory agents can be found, e.g., in U.S.Publication No. 2005/0002934 A1 at paragraphs 259-275 which isincorporated herein by reference in its entirety. In one embodiment, theimmunomodulatory agent is a chemotherapeutic agent. In an alternativeembodiment, the immunomodulatory agent is an immunomodulatory agentother than a chemotherapeutic agent. In some embodiments, thetherapy(ies) used in accordance with the invention is not animmunomodulatory agent.

In some embodiments, the therapy(ies) used in combination with acompound of the invention is an anti-angiogenic agent. Non-limitingexamples of anti-angiogenic agents include proteins, polypeptides,peptides, conjugates, antibodies (e.g., human, humanized, chimeric,monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab)2 fragments, andantigen-binding fragments thereof) such as antibodies that specificallybind to TNF-α, nucleic acid molecules (e.g., antisense molecules ortriple helices), organic molecules, inorganic molecules, and smallmolecules that reduce or inhibit angiogenesis. Other examples ofanti-angiogenic agents can be found, e.g., in U.S. Publication No.2005/0002934 A1 at paragraphs 277-282, which is incorporated byreference in its entirety. In other embodiments, the therapy(ies) usedin accordance with the invention is not an anti-angiogenic agent.

In some embodiments, the therapy(ies) used in combination with acompound of the invention is an anti-inflammatory agent. Non-limitingexamples of anti-inflammatory agents include any anti-inflammatoryagent, including agents useful in therapies for inflammatory disorders,well-known to one of skill in the art. Non-limiting examples ofanti-inflammatory agents include non-steroidal anti-inflammatory drugs(NSAIDs), steroidal anti-inflammatory drugs, anticholinergics (e.g.,atropine sulfate, atropine methylnitrate, and ipratropium bromide(ATROVENT™)), beta2-agonists (e.g., abuterol (VENTOLIN™ and PROVENTIL™),bitolterol (TORNALATE™), levalbuterol (XOPONEX™), metaproterenol(ALUPENT™), pirbuterol (MAXAIR™), terbutlaine (BRETHAIRE™ andBRETHINE™), albuterol (PROVENTIL™, REPETABS™, and VOLMAX™), formoterol(FORADIL AEROLIZER™), and salmeterol (SEREVENT™ and SEREVENT DISKUS™)),and methylxanthines (e.g., theophylline (UNIPHYL™ THEO-DUR™, SLO-BID™,AND TEHO-42TM)). Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxgenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™) corticosteroids (e.g., methylprednisolone (MEDROL™)),cortisone, hydrocortisone, prednisone (PREDNISONE™ and DELTASONE™),prednisolone (PRELONE™ and PEDIAPRED™), triamcinolone, azulfidine, andinhibitors of eicosanoids (e.g., prostaglandins, thromboxanes, andleukotrienes. Other examples of anti-inflammatory agents can be found,e.g., in U.S. Publication No. 005/0002934 A1 at paragraphs 290-294,which is incorporated by reference in its entirety. In otherembodiments, the therapy(ies) used in accordance with the invention isnot an anti-inflammatory agent.

In certain embodiments, the therapy(ies) used is an alkylating agent, anitrosourea, an antimetabolite, and anthracyclin, a topoisomerase IIinhibitor, or a mitotic inhibitor. Alkylating agents include, but arenot limited to, busulfan, cisplatin, carboplatin, cholormbucil,cyclophosphamide, ifosfamide, decarbazine, mechlorethamine, mephalen,and themozolomide. Nitrosoureas include, but are not limited tocarmustine (BCNU) and lomustine (CCNU). Antimetabolites include but arenot limited to 5-fluorouracil, capecitabine, methotrexate, gemcitabine,cytarabine, and fludarabine. Anthracyclins include but are not limitedto daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone.Topoisomerase II inhibitors include, but are not limited to, topotecan,irinotecan, etopiside (VP-16), and teniposide. Mitotic inhibitorsinclude, but are not limited to taxanes (paclitaxel, docetaxel), and thevinca alkaloids (vinblastine, vincristine, and vinorelbine).

The invention includes the use of agents that target cancer stem cellsin combination with a compound of the invention. In some embodiments,the agent used is an agent that binds to a marker, e.g., antigen oncancer stem cells. In a specific embodiment, the agent binds to anantigen that is expressed at a greater level on cancer stem cells thanon normal stem cells. In a specific embodiment, the agent bindsspecifically to a cancer stem cell antigen. In other embodiments, thetherapy(ies) used in accordance with the invention is an agent thatbinds to a marker on cancer stem cells. Non-limiting examples ofantigens on cancer stem cells that can be used to target cancer stemcells include CD34+/CD38−, CD34+/CD38−/CD123+, CD44+/CD24−, CD133+,CD34+/CD10−/CD19−, CD138−/CD34−/CD19+, CD20+, CD133+/RC2+,CD44+/α2β1hi/C133+, CD47+ and CD96+. In one embodiment, the agent thatbinds to a marker on cancer stem cells is an antibody. In anotherembodiment, the agent that binds to a marker on cancer stem cells is aligand. In certain embodiments, the antibody or ligand is attacheddirectly or indirectly to a therapeutic moiety. Non-limiting examples oftherapeutic moieties include, but are not limited to alkylating agents,anti-metabolites, plant alkaloids, cytotoxic agents, chemotherapeuticagents (e.g., a steroid, cytosine arabinoside, fluoruracil,methotrexate, aminopterin, mitomycin C, demecolcine, etoposide,mithramycin, calicheamicin, CC-1065, chlorambucil or melphalan),radionuclides, therapeutic enzymes, cytokines, toxins includingplant-derived toxins, fungus-derived toxins, bacteria-derived toxin(e.g., deglycosylated ricin A chain, a ribosome inactivating protein,alpha-sarcin, aspergillin, restirictocin, a ribonuclease, a diphtheriatoxin, Pseudomonas exotoxin, a bacterical endotoxin or the lipid Amoiety of a bacterial endotoxin), growth modulators and RNase.

For example, in a specific embodiment, the agent binds specifically tothe IL-3 Receptor (IL-3R). In some embodiments, the agent that binds tothe IL-3R is an antibody or an antibody fragment that is specific forIL-3R. In some embodiments, the antibody or antibody fragment isconjugated either chemically or via recombinant technology to atherapeutic moiety (e.g., a chemotherapeutic agent, a plant-, fungus- orbacteria-derived toxin, a radionuclide) using a linking agent to effecta cell killing response. In certain embodiments, the antibody,antibody-conjugate, antibody fragment, or antibody fragment-conjugatebinds to the a-subunit of IL-3R (i.e., the CD123 antigen). In otherembodiments, the antibody, antibody-conjugate, antibody fragment, orantibody fragment-conjugate binds to the IL-3R, containing both the αand β subunits. Methods for preparing antibodies to IL-3R and mimeticsof antibodies to IL-3R are described in U.S. Pat. No. 6,733,743 B2,which is incorporated herein by reference in its entirety.

In certain embodiments, antibodies or fragments that bind to a marker oncancer stem cells are substantially non-immunogenic in the treatedsubject. Non-immunogenic antibodies include, but are not limited to,chimerized antibodies, humanized antibodies, and antibodies from thesame species as the subject receiving the therapy. Antibodies orfragments that bind to markers in cancer stem cells can be producedusing techniques known in the art. See, for example, paragraphs 539-573of U.S. Publication No. 2005/0002934 A1, which is incorporated byreference in its entirety.

The invention includes the use of agents that target cancer stem cells.In certain embodiments, the agent acts alone. In other embodiments, theagent is attached directly or indirectly to another therapeutic moiety.Non-limiting examples of therapeutic moieties include, but are notlimited to, therapeutic enzymes, chemotherapeutic agents, cytokines,radionuclides, toxins, RNase, and antimetabolites. In some embodiments,the agent used is an agent that binds to a marker, e.g., an antigen on acancer stem cell. In a specific embodiment, the agent binds to anantigen that is expressed at a greater level on cancer stem cells thanon normal stem cells. In a specific embodiment, the agent binds to acancer stem cell antigen that is not a normal stem cell. In otherembodiments, the therapy(ies) is an agent that binds to a marker oncancer stem cells. In one embodiment, the agent that binds to a markeron cancer stem cells is an antibody, an antibody fragment, an antibodyconjugated to a therapeutic moiety, or an antibody, or an antibodyfragment conjugated to a therapeutic moiety.

In some embodiments, a compound of the invention is used in combinationwith radiation therapy comprising the use of X-rays, gamma rays andother sources of radiation to destroy cancer stem cells and/or cancercells. In specific embodiments, the radiation therapy is administered asexternal beam radiation or teletherapy, wherein the radiation isdirected from a remote source. In other embodiments, the radiationtherapy is administered as internal therapy or brachytherapy wherein aradioactive source is placed inside the body close to cancer stem cells,cancer cells and/or a tumor mass.

In some embodiments, the therapy used is a proliferation-based therapy.Non-limiting examples of such therapies include a chemotherapy andradiation therapy as described supra.

Currently available cancer therapies and their dosages, routes ofadministration and recommended usage are known in the art and have beendescribed in such literature as the Physician's Desk Reference (60thed., 2006). In accordance with the present invention, the dosages andfrequency of administration of chemotherapeutic agents are describedsupra.

5.9 Methods of Monitoring Cancer Stem Cells

As part of the therapeutically effective regimens of the invention, thecancer stem cell population can be monitored to assess the efficacy of atherapy as well as to determine prognosis of a subject with cancer orthe efficacy of a therapeutically effective regimen. In certainembodiments of the therapeutically effective therapies or regimens ofthe invention, the therapies or regimens result in a stabilization orreduction in the cancer stem cell population in the patient. In oneembodiment, the subject undergoing the regimen is monitored to assesswhether the regimen has resulted in a stabilization or reduction in thecancer stem cell population in the subject.

In some embodiments, the amount of cancer stem cells in a subject isdetermined using a technique well-known to one of skill in the art.

In accordance with the invention, cancer stem cells comprise a uniquesubpopulation (often 0.1-10% or so) of a tumor that, in contrast to theremaining 90% or so of the tumor (i.e., the tumor bulk), are relativelymore tumorigenic and relatively more slow-growing or quiescent. Giventhat conventional therapies and regimens have, in large part, beendesigned to attack rapidly proliferating cells (i.e., those cancer cellsthat comprise the tumor bulk), slower growing cancer stem cells may berelatively more resistant than faster growing tumor bulk to conventionaltherapies and regimens. This would explain another reason for thefailure of standard oncology treatment regimens to ensure long-termbenefit in most patients with advanced stage cancers. In a specificembodiment, a cancer stem cell(s) is the founder cell of a tumor (i.e.,it is the progenitor of cancer cells). In some embodiments, a cancerstem cell(s) has one, two, three, or more or all of the followingcharacteristics or properties: (i) can harbor the ability to initiate atumor and/or to perpetuate tumor growth, (ii) can be generallyrelatively less mutated than the bulk of a tumor (e.g. due to slowergrowth and thus fewer DNA replication-dependent errors, improved DNArepair, and/or epigenetic/non-mutagenic changes contributing to theirmalignancy), (iii) can have many features of a normal stem cell(s)(e.g., similar cell surface antigen and/or intracellular expressionprofile, self-renewal programs, multi-drug resistance, an immaturephenotype, etc., characteristic of normal stem cells) and may be derivedfrom a normal stem cell(s), (iv) can be potentially responsive to itsmicroenvironment (e.g., the cancer stem cells may be capable of beinginduced to differentiate and/or divide asymmetrically), (v) can be thesource of metastases, (vi) can be slow-growing or quiescent, (vii) canbe symmetrically-dividing, (viii) can be tumorigenic (e.g. as determinedby NOD/SCID implantation experiments), (ix) can be relatively resistantto traditional therapies (i.e. chemoresistant), and (x) can comprise asubpopulation of a tumor (e.g. relative to the tumor bulk).

In other embodiments, the amount of cancer stem cells in a sample from asubject is determined/assessed using a technique described herein orwell-known to one of skill in the art. Such samples include, but are notlimited to, biological samples and samples derived from a biologicalsample. In certain embodiments, in addition to the biological sampleitself or in addition to material derived from the biological samplesuch as cells, the sample used in the methods of this inventioncomprises added water, salts, glycerin, glucose, an antimicrobial agent,paraffin, a chemical stabilizing agent, heparin, an anticoagulant, or abuffering agent. In certain embodiments, the biological sample is blood,serum, urine, bone marrow or interstitial fluid. In another embodiment,the sample is a tissue sample. In a particular embodiment, the tissuesample is breast, brain, skin, colon, lung, liver, ovarian, pancreatic,prostate, renal, bone or skin tissue. In a specific embodiment, thetissue sample is a biopsy of normal or tumor tissue. The amount ofbiological sample taken from the subject will vary according to the typeof biological sample and the method of detection to be employed. In aparticular embodiment, the biological sample is blood, serum, urine, orbone marrow and the amount of blood, serum, urine, or bone marrow takenfrom the subject is 0.1 ml, 0.5 ml, 1 ml, 5 ml, 8 ml, 10 ml or more. Inanother embodiment, the biological sample is a tissue and the amount oftissue taken from the subject is less than 10 milligrams, less than 25milligrams, less than 50 milligrams, less than 1 gram, less than 5grams, less than 10 grams, less than 50 grams, or less than 100 grams.

In accordance with the methods of the invention, a sample derived from abiological sample is one in which the biological sample has beensubjected to one or more pretreatment steps prior to the detectionand/or measurement of the cancer stem cell population in the sample. Incertain embodiments, a biological fluid is pretreated by centrifugation,filtration, precipitation, dialysis, or chromatography, or by acombination of such pretreatment steps. In other embodiments, a tissuesample is pretreated by freezing, chemical fixation, paraffin embedding,dehydration, permeablization, or homogenization followed bycentrifugation, filtration, precipitation, dialysis, or chromatography,or by a combination of such pretreatment steps. In certain embodiments,the sample is pretreated by removing cells other than stem cells orcancer stem cells from the sample, or removing debris from the sampleprior to the determination of the amount of cancer stem cells in thesample according to the methods of the invention.

The samples for use in the methods of this invention may be taken fromany animal subject, preferably mammal, most preferably a human. Thesubject from which a sample is obtained and utilized in accordance withthe methods of this invention includes, without limitation, anasymptomatic subject, a subject manifesting or exhibiting 1, 2, 3, 4 ormore symptoms of cancer, a subject clinically diagnosed as havingcancer, a subject predisposed to cancer, a subject suspected of havingcancer, a subject undergoing therapy for cancer, a subject that has beenmedically determined to be free of cancer (e.g., following therapy forthe cancer), a subject that is managing cancer, or a subject that hasnot been diagnosed with cancer. In certain embodiments, the term “has nodetectable cancer” as used herein, refers to a subject or subjects inwhich there is no detectable cancer by conventional methods, e.g. MRI.In other embodiments, the term refers to a subject or subjects free fromany disorder.

In certain embodiments, the amount of cancer stem cells in a subject ora sample from a subject assessed prior to therapy or regimen (e.g. atbaseline) or at least 1, 2, 4, 6, 7, 8, 10, 12, 14, 15, 16, 18, 20, 30,60, 90 days, 6 months, 9 months, 12 months, >12 months after the subjectbegins receiving the therapy or regimen. In certain embodiments, theamount of cancer stem cells is assessed after a certain number of doses(e.g., after 2, 5, 10, 20, 30 or more doses of a therapy). In otherembodiments, the amount of cancer stem cells is assessed after 1 week, 2weeks, 1 month, 2 months, 1 year, 2 years, 3 years, 4 years or moreafter receiving one or more therapies.

In certain embodiments, a positive or negative control sample is asample that is obtained or derived from a corresponding tissue orbiological fluid or tumor as the sample to be analyzed in accordancewith the methods of the invention. This sample may come from the samepatient or different persons and at the same or different time points.

For clarity of disclosure, and not by way of limitation, the followingpertains to analysis of a blood sample from a patient. However, as oneskilled in the art will appreciate, the assays and techniques describedherein can be applied to other types of patient samples, including abody fluid (e.g. blood, bone marrow, plasma, urine, bile, asciticfluid), a tissue sample suspected of containing material derived from acancer (e.g. a biopsy) or homogenate thereof. The amount of sample to becollected will vary with the particular type of sample and method ofdetermining the amount of cancer stem cells used and will be an amountsufficient to detect the cancer stem cells in the sample.

A sample of blood may be obtained from a patient having differentdevelopmental or disease stages. Blood may be drawn from a subject fromany part of the body (e.g., a finger, a hand, a wrist, an arm, a leg, afoot, an ankle, a stomach, and a neck) using techniques known to one ofskill in the art, in particular methods of phlebotomy known in the art.In a specific embodiment, venous blood is obtained from a subject andutilized in accordance with the methods of the invention. In anotherembodiment, arterial blood is obtained and utilized in accordance withthe methods of the invention. The composition of venous blood variesaccording to the metabolic needs of the area of the body it isservicing. In contrast, the composition of arterial blood is consistentthroughout the body. For routine blood tests, venous blood is generallyused.

The amount of blood collected will vary depending upon the site ofcollection, the amount required for a method of the invention, and thecomfort of the subject. In some embodiments, any amount of blood iscollected that is sufficient to detect the amount of cancer stem cells.In a specific embodiment, lcc or more of blood is collected from asubject.

The amount of cancer stem cells in a sample can be expressed as thepercentage of, e.g., overall cells, overall cancer cells or overall stemcells in the sample, or quantitated relative to area (e.g. cells perhigh power field), or volume (e.g. cells per ml), or architecture (e.g.cells per bone spicule in a bone marrow specimen).

In some embodiments, the sample may be a blood sample, bone marrowsample, or a tissue/tumor biopsy sample, wherein the amount of cancerstem cells per unit of volume (e.g., 1 mL) or other measured unit (e.g.,per unit field in the case of a histological analysis) is quantitated.In certain embodiments, the cancer stem cell population is determined asa portion (e.g., a percentage) of the cancerous cells present in theblood or bone marrow or tissue/tumor biopsy sample or as a subset of thecancerous cells present in the blood or bone marrow or tissue/tumorbiopsy sample. The cancer stem cell population, in other embodiments,can be determined as a portion (e.g., percentage) of the total cells. Inyet other embodiments, the cancer stem cell population is determined asa portion (e.g., a percentage) of the total stem cells present in theblood sample.

In other embodiments, the sample from the patient is a tissue sample(e.g., a biopsy from a subject with or suspected of having canceroustissue), where the amount of cancer stem cells can be measured, forexample, by immunohistochemistry or flow cytometry, or on the basis ofthe amount of cancer stem cells per unit area, volume, or weight of thetissue. In certain embodiments, the cancer stem cell population (theamount of cancer stem cells) is determined as a portion (e.g., apercentage) of the cancerous cells present in the tissue sample or as asubset of the cancerous cells present in the tissue sample. In yet otherembodiments, the cancerous stem cell population (the amount of cancerstem cells) is determined as a portion (e.g., a percentage) of theoverall cells or stem cell cells in the tissue sample.

The amount of cancer stem cells in a test sample can be compared withthe amount of cancer stem cells in reference sample(s) to assess theefficacy of the regimen. In one embodiment, the reference sample is asample obtained from the subject undergoing therapy at an earlier timepoint (e.g., prior to receiving the regimen as a baseline referencesample, or at an earlier time point while receiving the therapy). Inthis embodiment, the therapy desirably results in a decrease in theamount of cancer stem cells in the test sample as compared with thereference sample. In another embodiment, the reference sample isobtained from a healthy, subject who has no detectable cancer, or from apatient that is in remission for the same type of cancer. In thisembodiment, the therapy desirably results in the test sample having anequal amount of cancer stem cells, or less than the amount of cancerstem cells than are detected in the reference sample.

In other embodiments, the cancer stem cell population in a test samplecan be compared with a predetermined reference range and/or a previouslydetected amount of cancer stem cells determined for the subject to gaugethe subject's response to the regimens described herein. In a specificembodiment, a stabilization or reduction in the amount of cancer stemcells relative to a predetermined reference range and/or earlier(previously detected) cancer stem cell amount determined for the subjectindicates an improvement in the subject's prognosis or a positiveresponse to the regimen, whereas an increase relative to thepredetermined reference range and/or earlier cancer stem cell amountindicates the same or worse prognosis, and/or a failure to respond tothe regimen. The cancer stem cell amount can be used in conjunction withother measures to assess the prognosis of the subject and/or theefficacy of the regimen. In a specific embodiment, the predeterminedreference range is based on the amount of cancer stem cells obtainedfrom a patient or population(s) of patients suffering from the same typeof cancer as the patient undergoing the therapy.

Generally, since stem cell antigens can be present on both cancer stemcells and normal stem cells, a sample from the cancer-afflicted patientwill have a higher stem cell count than a sample from a healthy, subjectwho has no detectable cancer due to the presence of the cancer stemcells. The therapy will desirably result in a cancer stem cell count forthe test sample (e.g., the sample from the patient undergoing therapy)that decreases and becomes increasingly closer to the stem cell count ina reference sample that is sample from a healthy, subject who has nodetectable cancer.

If the reduction in the amount of cancer stem cells is determined to beinadequate upon comparing the amount of cancer stem cells in the samplefrom the subject undergoing the regimen with the reference sample, thenthe medical practitioner has a number of possible options to adjust theregimen. For instance, the medical practitioner can then increase eitherthe dosage or intensity of the therapy administered, the frequency ofthe administration, the duration of administration, combine the therapywith another therapy(ies), change the management altogether includinghalting therapy, or any combination thereof.

In certain embodiments, the dosage, frequency and/or duration ofadministration of a therapy is modified as a result of the change in theamount of cancer stem cells detected in or from the treated patient. Forexample, if a subject receiving therapy for leukemia has a cancer stemcell measurement of 2.5% of his tumor prior to therapy and 5% after 6weeks of therapy, then the therapy or regimen may be altered or stoppedbecause the increase in the percentage of cancer stem cells indicatesthat the therapy or regimen is not optimal. Alternatively, if anothersubject with leukemia has a cancer stem cell measurement of 2.5% of histumor prior to therapy and 1% after 6 weeks of therapy, then the therapyor regimen may be continued because the decrease in the percentage ofcancer stem cells indicates that the therapy or regimen is effective.

The amount of cancer stem cells can be monitored/assessed using standardtechniques known to one of skill in the art. Cancer stem cells can bemonitored by, e.g., obtaining a sample, such as a tissue/tumor sample,blood sample or a bone marrow sample, from a subject and detectingcancer stem cells in the sample. The amount of cancer stem cells in asample (which may be expressed as percentages of, e.g., overall cells oroverall cancer cells) can be assessed by detecting the expression ofantigens on cancer stem cells. Techniques known to those skilled in theart can be used for measuring these activities. Antigen expression canbe assayed, for example, by immunoassays including, but not limited to,western blots, immunohistochemistry, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, immunofluorescence, protein A immunoassays, flowcytometry, and FACS analysis. In such circumstances, the amount ofcancer stem cells in a test sample from a subject may be determined bycomparing the results to the amount of stem cells in a reference sample(e.g., a sample from a subject who has no detectable cancer) or to apredetermined reference range, or to the patient him/herself at anearlier time point (e.g. prior to, or during therapy).

In a specific embodiment, the cancer stem cell population in a samplefrom a patient is determined by flow cytometry. This method exploits thedifferential expression of certain surface markers on cancer stem cellsrelative to the bulk of the tumor. Labeled antibodies (e.g., fluorescentantibodies) can be used to react with the cells in the sample, and thecells are subsequently sorted by FACS or flow cytometry methods. In someembodiments, a combination of cell surface markers are utilized in orderto determine the amount of cancer stem cells in the sample. For example,both positive and negative cell sorting may be used to assess the amountof cancer stem cells in the sample. Cancer stem cells for specific tumortypes can be determined by assessing the expression of markers on cancerstem cells. In certain embodiments, the tumors harbor cancer stem cellsand their associated markers as set forth in Table 2 below, whichprovides a non-limiting list of cancer stem cell phenotypes associatedwith various types of cancer.

TABLE 2 Tumor Cancer Stem Cell Phenotype Leukemia (AML) CD34+/CD38−Breast CD44+/CD24− Brain CD133+ Leukemia (ALL) CD34+/CD10−/CD19− OvarianCD44+/CD24− Multiple Myeloma CD138−/CD34−/CD19+ Chronic myelogenousleukemia CD34+/CD38− Melanoma CD20+ Ependymoma CD133+/RC2+ ProstateCD44+/α₂β₁ ^(hi)/CD133+

Additional cancer stem cell markers include, but are not limited to,CD123, CLL-1, combinations of SLAMs (signaling lymphocyte activationmolecule family receptors; see Yilmaz, et al., Hematopoiesis 107:924-930 (2006)), such as CD150, CD244, and CD48, and those markersdisclosed in U.S. Pat. No. 6,004,528 to Bergstein, in pending U.S.patent application Ser. No. 09/468,286, and in U.S. Patent ApplicationPublication Nos. 2006/0083682, 2007/0036800, 2007/0036801, 2007/0036802,2007/0041984, 2007/0036803, and 2007/0036804, each of which areincorporated herein by reference in their entirety. See, e.g., Table 1of U.S. Pat. No. 6,004,528 and Tables 1, 2, and 3 of U.S. patentapplication Ser. No. 09/468,286 and U.S. Patent Application PublicationNos. 2006/0083682, 2007/0036800, 2007/0036801, 2007/0036802,2007/0041984, 2007/0036803, and 2007/0036804.

In a specific embodiment the cancer stem population in a sample, e.g., atissue sample, such as a solid tumor biopsy, is determined usingimmunohistochemistry techniques. This method exploits the differentialexpression of certain surface markers on cancer stem cells relative tothe bulk of the tumor. Labeled antibodies (e.g., fluorescent antibodies)can be used to react with the cells in the sample, and the tissue issubsequently stained. In some embodiments, a combination of certain cellsurface markers are utilized in order to determine the amount of cancerstem cells in the sample. Cancer stem cells for specific tumor types canbe determined by assessing the expression of certain markers that arespecific to cancer stem cells. In certain embodiments, the tumors harborcancer stem cells and their associated markers as set forth in Table 2above.

Suitable cancer stem cell antigens may be identified: (i) throughpublicly available information, such as published and unpublishedexpression profiles including cell surface antigens of cancer stem cellsof a particular tumor type or adult stem cells for a particular tissuetype (e.g. Table 2), and/or (ii) by cloning cancer stem cells or adultstem cells of a particular tumor or tissue type, respectively, in orderto determine their expression profiles and complement of cell surfaceantigens. Cloning of normal stem cells is a technique routinely employedin the art (Uchida, et al., Curr Opin Immunol, 5:177-184 (1993)). Infact, this same technique is used to identify normal stem cells andcancer stem cells. Moreover, assumption that a proportion of normal stemcell gene products, e.g. cell surface antigens, will also be present oncancer stem cells derived from the same tissue type has proven aneffective way to identify cancer stem cell gene products and cancer stemcells. For example, knowledge that the normal hematopoietic stem cellwas CD34+/CD38− resulted in the determination that acute myeloidleukemia (AML) stem cells is similarly CD34+/CD38−. This indeed wasconfirmed by standard stem cell cloning techniques (See Bonnet, et al.,Nat Med 3:730-737 (1997)). Brain cancer stem cells were similarlyisolated using a marker of normal (brain) stem cells, in this case CD133(See Singh, et al., Nature 432(7015):396-401 (2004)).

In certain embodiments using flow cytometry of a sample, the Hoechst dyeprotocol can be used to identify cancer stem cells in tumors. Briefly,two Hoechst dyes of different colors (typically red and blue) areincubated with tumor cells. The cancer stem cells, in comparison withbulk cancer cells, over-express dye efflux pumps on their surface thatallow these cells to pump the dye back out of the cell. Bulk tumor cellslargely have fewer of these pumps, and are therefore relatively positivefor the dye, which can be detected by flow cytometry. Typically agradient of dye positive (“dye⁺”) vs. dye negative (“dye⁻”) cellsemerges when the entire population of cells is observed. Cancer stemcells are contained in the dye⁻ or dye low (dye^(low)) population. Foran example of the use of the Hoechst dye protocol to characterize a stemcell or cancer stem cell population see Goodell, et al., Blood,98(4):1166-1173 (2001) and Kondo, et al., Proc Natl Acad Sci USA101:781-786 (2004). In this way, flow cytometry could be used to measurecancer stem cell amount pre- and post-therapy to assess the change incancer stem cell amount arising from a given therapy or regimen.

In other embodiments using flow cytometry of a sample, the cells in thesample may be treated with a substrate for aldehyde dehydogenase thatbecomes fluorescent when catalyzed by this enzyme. For instance, thesample can be treated with BODIPY®—aminoacetaldehyde which iscommercially available from StemCell Technologies Inc. as Aldefluor®.Cancer stem cells express high levels of aldehyde dehydrogenase relativeto bulk cancer cells and therefore become brightly fluorescent uponreaction with the substrate. The cancer stem cells, which becomefluorescent in this type of experiment, can then be detected and countedusing a standard flow cytometer. In this way, flow cytometry could beused to measure cancer stem cell amount pre- and post-therapy to assessthe change in cancer stem cell amount arising from a given therapy orregimen.

In other embodiments, a sample (e.g., a tumor or normal tissue sample,blood sample or bone marrow sample) obtained from the patient iscultured in in vitro systems to assess the cancer stem cell populationor amount of cancer stem cells. For example, tumor samples can becultured on soft agar, and the amount of cancer stem cells can becorrelated to the ability of the sample to generate colonies of cellsthat can be visually counted. Colony formation is considered a surrogatemeasure of stem cell content, and thus, can be used to quantitate theamount of cancer stem cells. For instance, with hematological cancers,colony-forming assays include colony forming cell (CFC) assays,long-term culture initiating cell (LTC-IC) assays, and suspensionculture initiating cell (SC-IC) assays. In this way, the colony-formingor related assay could be used to measure cancer stem cell amount pre-and post-therapy to assess the change in cancer stem cell amount arisingfrom a given therapy or regimen.

In other embodiments, sphere formation is measured to determine theamount of cancer stem cells in a sample (e.g., cancer stem cells formthree-dimensional clusters of cells, called spheres) in appropriatemedia that is conducive to forming spheres. Spheres can be quantitatedto provide a measure of cancer stem cells. See Singh, et al., Cancer Res63: 5821-5828 (2003). Secondary spheres can also be measured. Secondaryspheres are generated when the spheres that form from the patient sampleare broken apart, and then allowed to reform. In this way, thesphere-forming assay could be used to measure cancer stem cell amountpre- and post-therapy to assess the change in cancer stem cell amountarising from a given therapy or regimen.

In other embodiments, the amount of cancer stem cells in a sample can bedetermined with a cobblestone assay. Cancer stem cells from certainhematological cancers form “cobblestone areas” (CAs) when added to aculture containing a monolayer of bone marrow stromal cells. Forinstance, the amount of cancer stem cells from a leukemia sample can beassessed by this technique. The tumor samples are added to the monolayerof bone marrow stromal cells. The leukemia cancer stem cells, more sothan the bulk leukemia cells, have the ability to migrate under thestromal layer and seed the formation of a colony of cells which can beseen visually under phase contrast microscopy in approximately 10-14days as CAs. The number of CAs in the culture is a reflection of theleukemia cancer stem cell content of the tumor sample, and is considereda surrogate measure of the amount of stem cells capable of engraftingthe bone marrow of immunodeficient mice. This assay can also be modifiedso that the CAs can be quantitated using biochemical labels ofproliferating cells instead of manual counting, in order to increase thethroughput of the assay. See Chung, et al., Blood 105(1):77-84 (2005).In this way, the cobblestone assay could be used to measure cancer stemcell amount pre- and post-therapy to assess the change in cancer stemcell amount arising from a given therapy or regimen.

In other embodiments, a sample (e.g., a tumor or normal tissue sample,blood sample or bone marrow sample) obtained from the patient isanalyzed in in vivo systems to determine the cancer stem cell populationor amount of cancer stem cells. In certain embodiments, for example, invivo engraftment is used to quantitate the amount of cancer stem cellsin a sample. In vivo engraftment involves implantation of a humanspecimen with the readout being the formation of tumors in an animalsuch as in immunocompromised or immunodeficient mice (such as NOD/SCIDmice). Typically, the patient sample is cultured or manipulated in vitroand then injected into the mice. In these assays, mice can be injectedwith a decreasing amount of cells from patient samples, and thefrequency of tumor formation can be plotted vs. the amount of cellsinjected to determine the amount of cancer stem cells in the sample.Alternatively, the rate of growth of the resulting tumor can bemeasured, with larger or more rapidly advancing tumors indicating ahigher cancer stem cell amount in the patient sample. In this way, an invivo engraftment model/assay could be used to measure cancer stem cellamount pre- and post-therapy to assess the change in cancer stem cellamount arising from a given therapy or regimen.

In certain in vivo techniques, an imaging agent, or diagnostic moiety,is used which binds to molecules on cancer cells or cancer stem cells,e.g., cancer cell or cancer stem cell surface antigens. For instance, afluorescent tag, radionuclide, heavy metal, or photon-emitter isattached to an antibody (including an antibody fragment) that binds to acancer stem cell surface antigen. Exemplary cancer stem cell surfaceantigens are listed above in Table 2. The medical practitioner caninfuse the labeled antibody into the patient either prior to, during, orfollowing treatment, and then the practitioner can place the patientinto a total body scanner/developer which can detect the attached label(e.g., fluorescent tag, radionuclide, heavy metal, photon-emitter). Thescanner/developer (e.g., CT, MRI, or other scanner, e.g. detector offluorescent label, that can detect the label) records the presence,amount/quantity, and bodily location of the bound antibody. In thismanner, the mapping and quantitation of tag (e.g. fluorescence,radioactivity, etc.) in patterns (i.e., different from patterns ofnormal stem cells within a tissue) within a tissue or tissues indicatesthe treatment efficacy within the patient's body when compared to areference control such as the same patient at an earlier time point or apatient or healthy individual who has no detectable cancer. For example,a large signal (relative to a reference range or a prior treatment date,or prior to treatment) at a particular location indicates the presenceof cancer stem cells. If this signal is increased relative to a priordate it suggests a worsening of the disease and failure of therapy orregimen. Alternatively, a signal decrease indicates that the therapy orregimen has been effective.

In a specific embodiment, the amount of cancer stem cells is detected invivo in a subject according to a method comprising the steps of: (a)administering to the subject an effective amount of a labeled cancerstem cell marker binding agent that binds to a cell surface marker foundon the cancer stem cells, and (b) detecting the labeled agent in thesubject following a time interval sufficient to allow the labeled agentto concentrate at sites in the subject where the cancer stem cellsurface marker is expressed. In accordance with this embodiment, thecancer stem cell surface marker-binding agent is administered to thesubject according to any suitable method in the art, for example,parenterally (such as intravenously), or intraperitoneally. Inaccordance with this embodiment, the effective amount of the agent isthe amount which permits the detection of the agent in the subject. Thisamount will vary according to the particular subject, the label used,and the detection method employed. For example, it is understood in theart that the size of the subject and the imaging system used willdetermine the amount of labeled agent needed to detect the agent in asubject using an imaging means. In the case of a radiolabeled agent fora human subject, the amount of labeled agent administered is measured interms of radioactivity, for example from about 5 to 20 millicuries of⁹⁹Tc. The time interval following the administration of the labeledagent which is sufficient to allow the labeled agent to concentrate atsites in the subject where the cancer stem cell surface marker isexpressed will vary depending on several factors, for example, the typeof label used, the mode of administration, and the part of the subject'sbody that is imaged. In a particular embodiment, the time interval thatis sufficient is 6 to 48 hours, 6 to 24 hours, or 6 to 12 hours. Inanother embodiment the time interval is 5 to 20 days or 5 to 10 days.The presence of the labeled cancer stem cell surface marker-bindingagent can be detected in the subject using imaging means known in theart. In general, the imaging means employed depend upon the type oflabel used. Skilled artisans will be able to determine the appropriatemeans for detecting a particular label. Methods and devices that may beused include, but are not limited to, computed tomography (CT), wholebody scan such as position emission tomography (PET), magnetic resonanceimaging (MRI), and sonography. In a specific embodiment, the cancer stemcell surface marker-binding agent is labeled with a radioisotope and isdetected in the patient using a radiation responsive surgical instrument(Thurston, et al., U.S. Pat. No. 5,441,050). In another embodiment, thecancer stem cell surface marker-binding agent is labeled with afluorescent compound and is detected in the patient using a fluorescenceresponsive scanning instrument. In another embodiment, the cancer stemcell surface marker-binding agent is labeled with a positron emittingmetal and is detected in the patient using positron emission-tomography.In yet another embodiment, the cancer stem cell surface marker-bindingagent is labeled with a paramagnetic label and is detected in a patientusing magnetic resonance imaging (MRI).

Any in vitro or in vivo (ex vivo) assays known to those skilled in theart that can detect and/or quantify cancer stem cells can be used tomonitor cancer stem cells in order to evaluate the prophylactic and/ortherapeutic utility of a cancer therapy or regimen disclosed herein forcancer or one or more symptoms thereof; or these assays can be used toassess the prognosis of a patient. The results of these assays then maybe used to possibly maintain or alter the cancer therapy or regimen.

The amount of cancer stem cells in a specimen can be compared to apredetermined reference range and/or an earlier amount of cancer stemcells previously determined for the subject (either prior to, or duringtherapy) in order to gauge the subject's response to the treatmentregimens described herein. In a specific embodiment, a stabilization orreduction in the amount of cancer stem cells relative to a predeterminedreference range and/or earlier cancer stem cell amount previouslydetermined for the subject (prior to, during and/or after therapy)indicates that the therapy or regimen was effective and thus possibly animprovement in the subject's prognosis, whereas an increase relative tothe predetermined reference range and/or cancer stem cell amountdetected at an earlier time point indicates that the therapy or regimenwas ineffective and thus possibly the same or a worsening in thesubject's prognosis. The cancer stem cell amount can be used with otherstandard measures of cancer to assess the prognosis of the subjectand/or efficacy of the therapy or regimen: such as response rate,durability of response, relapse-free survival, disease-free survival,progression-free survival, and overall survival. In certain embodiments,the dosage, frequency and/or duration of administration of a therapy ismodified as a result of the determination of the amount or change inrelative amount of cancer stem cells at various time points which mayinclude prior to, during, and/or following therapy.

The present invention also relates to methods for determining that acancer therapy or regimen is effective at targeting and/or impairingcancer stem cells by virtue of monitoring cancer stem cells over timeand detecting a stabilization or decrease in the amount of cancer stemcells during and/or following the course of the cancer therapy orregimen.

In a certain embodiment, a therapy or regimen may be marketed as ananti-cancer stem cell therapy or regimen based on the determination thata therapy or regimen is effective at targeting and/or impairing cancerstem cells by virtue of having monitored or detected a stabilization ordecrease in the amount of cancer stem cells during therapy.

5.10 Methods of Monitoring Cancer Cells

As part of the therapeutically effective regimens of the invention, theamount of cancer cells (alone or in combination with the amount ofcancer stem cells) can be monitored/assessed using standard techniquesknown to one of skill in the art. In certain embodiments of thetherapeutically effective regimens of the invention, the regimens resultin a stabilization or reduction in the amount (expressed, e.g., as apercentage) of cancer cells in the subject. In one embodiment, thesubject undergoing the regimen is monitored to determine whether theregimen has resulted in a stabilization or reduction in the amount(expressed, e.g., as a percentage) of cancer cells in the subject.

In some embodiments, the amount of cancer cells is assessed in a subjectusing techniques described herein or known to one of skill in the art.In other embodiments, the amount of cancer cells is detected in asample. Such samples include, but are not limited to, biological samplesand samples derived from a biological sample. In certain embodiments, inaddition to the biological sample itself or in addition to materialderived from the biological sample such as cells, the sample used in themethods of this invention comprises added water, salts, glycerin,glucose, an antimicrobial agent, paraffin, a chemical stabilizing agent,heparin, an anticoagulant, or a buffering agent. In certain embodiments,the biological sample is blood, serum, urine, bone marrow orinterstitial fluid. In another embodiment, the sample is a tissuesample. In a particular embodiment, the tissue sample is breast, colon,lung, liver, ovarian, pancreatic, prostate, renal, bone or skin tissue.In a specific embodiment, the tissue sample is a biopsy, including atumor biopsy. The amount of biological sample taken from the subjectwill vary according to the type of biological sample and the method ofdetection to be employed. In a particular embodiment, the biologicalsample is blood, serum, or urine and the amount of blood, serum, orurine taken from the subject is 0.1 ml, 0.5 ml, 1 ml, 5 ml, 10 ml ormore. In another embodiment, the biological sample is a tissue and theamount of tissue taken from the subject is less than 10 milligrams, lessthan 25 milligrams, less than 50 milligrams, less than 1 gram, less than5 grams, less than 10 grams, less than 50 grams, or less than 100 grams.

In accordance with the methods of the invention, a sample derived from abiological sample is one in which the biological sample has beensubjected to one or more pretreatment steps prior to the detectionand/or measurement of the cancer cell population in the sample. Incertain embodiments, a biological fluid is pretreated by centrifugation,filtration, precipitation, dialysis, or chromatography, or by acombination of such pretreatment steps. In other embodiments, a tissuesample is pretreated by freezing, chemical fixation, paraffin embedding,dehydration, permeablization, or homogenization followed bycentrifugation, filtration, precipitation, dialysis, or chromatography,or by a combination of such pretreatment steps. In certain embodiments,the sample is pretreated by removing cells other than cancer cells fromthe sample, or removing debris from the sample prior to thedetermination of the amount of cancer cells in the sample according tothe methods of the invention.

The samples for use in the methods of this invention may be taken fromany animal subject, preferably a mammal, most preferably a human. Thesubject from which a sample is obtained and utilized in accordance withthe methods of this invention includes, without limitation, anasymptomatic subject, a subject manifesting or exhibiting 1, 2, 3, 4 ormore symptoms of cancer, a subject clinically diagnosed as havingcancer, a subject predisposed to cancer, a subject suspected of havingcancer, a subject undergoing therapy for cancer, a subject that has beenmedically determined to be free of cancer (e.g., following therapy forthe cancer), a subject that is managing cancer, or a subject that hasnot been diagnosed with cancer.

In certain embodiments, the amount of cancer cells is assessed in asubject or a sample from a subject at least 1, 2, 4, 6, 8, 10, 12, 14,15, 16, 18, 20, or 30, 60, 90 days 6 months, 9 months, 12 months, >12months after the subject begins receiving the regimen. In certainembodiments, the amount of cancer cells is assessed after a number ofdoses (e.g., after 1, 2, 5, 10, 20, 30 or more doses of a therapy). Inother embodiments, the amount of cancer cells is assessed after 2 weeks,1 month, 2 months, 1 year, 2 years, 3 years, 4 years or more afterreceiving one or more therapies.

The amount of cancer cells in a sample can be expressed as thepercentage of, e.g., overall cells in the sample. In some embodiments,the sample is a blood sample or bone marrow sample, wherein the amountof cancer cells per unit of volume (e.g., 1 mL) or other measured unit(e.g., per unit field in the case of a histological analysis) isquantitated. The cancer cell population, in certain embodiments, can bedetermined as a percentage of the total blood cells.

In other embodiments, the sample from the patient is a tissue sample(e.g., a biopsy from a subject with or suspected or having canceroustissue), where the amount of cancer cells can be measured, for example,by immunohistochemistry or on the basis of the amount of cancer cellsper unit weight of the tissue.

The amount of cancer cells in the test sample can be compared with theamount of cancer cells measured in a reference sample(s) to assess theefficacy of the regimen. In one embodiment, the reference sample is asample from the subject undergoing therapy, at an earlier time point(e.g., prior to receiving the regimen as a baseline reference sample, orat an earlier time point while receiving the therapy). In thisembodiment, the therapy desirably results in a decrease in the amount ofcancer cells in the test sample as compared with the reference sample.In another embodiment, the reference sample is obtained from a healthy,subject who has no detectable cancer, or from a patient that is inremission for the same type of cancer. In this embodiment, the therapydesirably results in the test sample having an equal amount of cancercells as detected in the reference sample (e.g., no detectable cancercells).

If the reduction in the amount of cancer cells is judged too small, thenthe medical practitioner has a number of options to adjust the regimen.For instance, the medical practitioner can then either increase thedosage of the therapy administered, the frequency of the administration,the duration of administration, combine the therapy with anothertherapy(ies), halt the therapy, or any combination thereof.

The amount of cancer cells can be monitored/assessed using standardtechniques known to one of skill in the art. Cancer cells can bemonitored by, e.g., obtaining a sample, such as a tumor sample, bloodsample or bone marrow sample, from a subject and detecting cancer cellsin the sample. The amount of cancer cells in a sample (which may beexpressed as a percentage) can be assessed by detecting the expressionof antigens on cancer cells and/or by detecting the proliferation ofcancer cells. Techniques known to those of skilled in the art can beused for measuring these activities. For example, cellular proliferationcan be assayed by 3H-thymidine incorporation assays and trypan blue cellcounts. Antigen expression can be assayed, for example, by immunoassaysincluding, but are not limited to western blots, immunohistochemistryradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, fluorescence-activatedcell sorter (FACS) analysis, flow cytometry and immunofluorescence.

The amount of cancer cells can be compared to a predetermined referencerange and/or an earlier amount of cancer cells determined for thesubject to gauge the subject's response to the regimens describedherein. In a specific embodiment, a reduction in the amount of cancercells relative to a predetermined reference range and/or earlier cancercell amount determined for the subject indicate an improvement in thesubject's prognosis or response to a therapy, whereas an increaserelative to the predetermined reference range and/or earlier cancer cellnumbers indicates the same or worse prognosis, or failure to respond toa therapy. In certain embodiments, the dosage, frequency and/or durationof administration of a therapy is modified as a result of the change inthe amount of cancer cells.

In some embodiments, the cancer cell population can bemonitored/assessed using gross measurements of the cancer cellpopulation. For example, in some embodiments, the cancer cell populationis determined using imaging methods such as computed tomography (CT),magnetic resonance imaging (MRI), ultrasound, X-ray imaging,mammography, radionuclide imaging, PET scan, palpitation, directmeasurement (e.g. with a ruler) or bone scans.

In embodiments of the invention comprising treatment of solid tumors,the bulk size of the tumor may provide an estimate of the cancer cellpopulation. A number of known methods can be used to assess the bulksize of the tumor. Non-limiting examples of such methods include imagingmethods (e.g., computed tomography (CT), magnetic resonance imaging(MRI), PET scans, palpitation, direct measurement (e.g. with a ruler),ultrasound, X-ray imaging, mammography, bone scans and radioisotopeimaging), visual methods (e.g., colonoscopy, bronchoscopy, endoscopy),physical examination (e.g., prostate examination, breast examination,lymph nodes examination, abdominal examination, general palpation),blood tests (e.g., prostate specific antigen (PSA) test,carcinoembryonic antigen (CEA) test, cancer antigen (CA)-125 test,alpha-fetoprotein (AFP)), bone marrow analyses (e.g., in cases ofhematological malignancies), histopathology, cytology and flowcytometry.

In some embodiments, the bulk tumor size can be measured by assessmentsbased on the size of tumor lesions determined from imaging methods. Inspecific embodiments, the assessments are performed in accordance withthe Response Evaluation Criteria In Solid Tumors (RECIST) Guidelines,which are set forth in Therasse, et al., J Nat Canc Inst 92(3), 205-216(2000). For instance, in specific embodiments, lesions in the subjectthat are representative of bulk tumor size are selected so that they areat least=20 mm in their longest diameter at baseline (prior totreatment) when conventional imaging techniques are used (e.g.,conventional CT scan, MRI or x-ray) and lesions that are at least =10 mmin their longest diameter at baseline should be selected when spiral CTscanning is used.

5.11 Methods of Monitoring Lymphocyte Cell Count, Neutrophil Cell Count,Platelet Count and Hemoglobin

As part of the prophylactically and/or therapeutically effectiveregimens of the invention, peripheral blood lymphocyte counts can bemonitered/assessed using standard techniques known to one of skill inthe art. Peripheral blood lymphocyte counts in a subject can bedetermined by, e.g., obtaining a sample of peripheral blood from saidsubject, separating the lymphocytes from other components of peripheralblood such as plasma using, e.g., Ficoll-Hypaque (Pharmacia) gradientcentrifugation, and counting the lymphocytes using trypan blue.Peripheral blood T-cell counts in a subject can be determined by, e.g.,separating the lymphocytes from other components of peripheral bloodsuch as plasma using, e.g., Ficoll-Hypaque (Pharmacia) gradientcentrifugation. Labeling the T-cells with an antibody directed to aT-cell antigen such as CD3, CD4, and CD8 which is conjugated to a FACSdetectable agent, such as FITC or phycoerythrin, and measuring theamount of T-cells by FACS. Further, the effect on a particular subset ofT cells (e.g., CD2+, CD4+, CD8+, CD45+, CD45RO+, CD45RA+, or CD8+RA+) orNK cells can be determined using standard techniques known to one ofskill in the art, such as FACS.

The subject's absolute neutrophil count (ANC) can be monitored/assessedusing standard techniques known to one of skill in the art. In someembodiments, the regimen includes monitoring the patient's ANC in orderto avoid the risk of the patient developing neutropenia.

The ANC can be calculated from measurements of the total number of whiteblood cells (WBC) and the numbers of neutrophils and bands (immatureneutrophils). The ANC can be determined manually by trained medicaltechnologists or by automated ANC results obtained from automatedhematology analyzers.

The subject's platelet count (PLT) can be monitored/assessed usingstandard techniques known to one of skill in the art. In someembodiments, the regimen includes monitoring the patient's plateletcount in order to avoid the risk of the patient developingthrombocytopenia or becoming blood transfusion dependent. Transfusionscan be given as determined by the physician.

The subject's hemoglobin (Hgb) can be monitored/assessed using standardtechniques known to one of skill in the art. In some embodiments, theregimen includes monitoring the patient's hemoglobin in order to avoidthe risk of the patient developing anemia or becoming transfusiondependent. Transfusions or growth factors (e.g. erythropoietin) can begiven as determined by the physician.

5.12 Biological Assays

5.12.1 In Vitro Assays

Antibodies and antibody conjugates of the invention may be characterizedin a variety of ways well-known to one of skill in the art. Inparticular, antibodies or conjugates of the invention may be assayed forthe ability to bind to the IL3R alpha chain. Such an assay may beperformed in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-421),on beads (Lam, 1991, Nature 354:82-84), on chips (Fodor, 1993, Nature364:555-556), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S.Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull etal., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scottand Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406;Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; andFelici, 1991, J. Mol. Biol. 222:301-310) (each of these references isincorporated herein in its entirety by reference). Antibodies orconjugates that have been identified to bind to the IL3R alpha chain ora fragment thereof can then be assayed for their specificity andaffinity for the IL3R alpha chain.

The antibodies or conjugates may be assayed for specific binding to theIL3R alpha chain and cross-reactivity with other antigens by any methodknown in the art. Immunoassays which can be used to analyze specificbinding and cross-reactivity include, but are not limited to,competitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew. Such assays are routine and well known in the art (see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York, which is incorporated by referenceherein in its entirety). Exemplary immunoassays are described brieflybelow (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 MaCl, 0.01 M sodium phosphate atpH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody or conjugate of interest to the cell lysate,incubating for a period of time (e.g., 1 to 4 hours) at 40 C, addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 40 C, washing the beads in lysisbuffer and resuspending the beads in SDS/sample buffer. The ability ofthe antibody or conjugate of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (e.g., the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of an antibody of the present invention for the IL3R alphachain and the binding off-rates can be determined from the data byscatchard plot analysis. Competition with a second antibody can also bedetermined using radioimmunoassays. In this case, the IL3R alpha chainis incubated with an antibody of the present invention conjugated to alabeled compound (e.g., ³H or ¹²⁵I) in the presence of increasingamounts of an unlabeled second antibody.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibodies or conjugates to the IL3Ralpha chain. BIAcore kinetic analysis comprises analyzing the bindingand dissociation of the IL3R alpha chain from chips with immobilizedantibodies or conjugates on their surface.

The antibodies, conjugates, pharmaceutical compositions and regimens ofthe invention can be tested in vitro and/or in vivo for their ability toreduce the quantity, number, amount or percentage of cancer cells and/orcancer stem cells, or inhibit their proliferation. The ability of aconjugate or a regimen of the invention to reduce the quantity, number,amount or percentage of cancer cells, cancer stem cells and/or immunecells (e.g., lymphocytes) or inhibit their proliferation can be assessedby: detecting the expression of antigens on cancer cells, cancer stemcells, and immune cells; detecting the proliferation or viability ofcancer cells, cancer stem cells and immune cells; detecting the effectorfunction of cancer cells and cancer stem cells. Techniques known tothose of skilled in the art can be used for measuring these activities.For example, cellular proliferation can be assayed by ³H-thymidineincorporation assays and trypan blue cell counts. Antigen expression canbe assayed, for example, by immunoassays including, but are not limitedto, competitive and non-competitive assay systems using techniques suchas western blots, immunohistochemistry radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, flow cytometry and FACS analysis.

An antibody, antibody conjugate, pharmaceutical composition, or regimenof the invention is preferably tested in vitro and then in vivo for thedesired therapeutic or prophylactic activity prior to use in humans. Forexample, assays which can be used to determine whether administration ofa specific antibody or antibody conjugate is indicated include cellculture assays in which a patient tissue sample (e.g., a cancer cell orcancer stem cell) is grown in culture and exposed to, or otherwisecontacted with, an antibody or antibody conjugate of the invention, andthe effect of such antibody or antibody conjugate upon the tissue sampleis observed. The tissue sample can be obtained by biopsy from thepatient. This test allows the identification of the therapeutically mosteffective therapy (e.g., prophylactic or therapeutic agent) for eachindividual patient.

Determination of cell viability using the XTT assay: In some cases,CD34+ cells are isolated from human cord blood using magnetic beadscoated with anti-CD34 antibody. Isolated cells are then counted andaliquoted into 96-well plates and then incubated in the presence ofvarying concentrations of an antibody or antibody conjugate of theinvention. Cell viability it measured by the addition of the XTTcolorimetric reagent. Viability is determined by the absorbance oftreated cultures at approximately 450-500 nm compared to untreatedcultures. In other cases, the cells used in the assay may be a leukemiacell line, such as MV4; 11. The assay can also be used to determine thetime course of cell killing by various compounds by performing the XTTassay on cultures that are incubated with the antibody or antibodyconjugate for varying periods of time.

Cobblestone assay: The cobblestone area-forming cell (CAFC) assayexploits a reproducible visual end point for the quantitation of cancerstem cells. Leukemia samples are added to adherent cultures of stromalcells, some embodiments MS-5 stromal cells. The cancer stem cells in theculture will migrate below the MS-5 stromal cells and form a colony ofcells called a cobblestone that can be visual quantitated. To test theeffect of an antibody or antibody conjugate of the invention on thecancer stem cell population using this assay, cells are first culturedin the presence of the drug. In some embodiments the cells are culturedfor 16 hours. After this incubation, the cells are added to the stromalcultures. A reduction in the cobblestone area formation in cultures thatwere treated with the drug compared to the untreated cells representscancer stem cell activity for the drug.

5.12.2 In Vivo Assays

The antibodies, antibody conjugates, pharmaceutical compositions, andregimens of the invention can be tested in suitable animal model systemsprior to use in humans. Such animal model systems include, but are notlimited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits,etc. Any animal system well-known in the art may be used. Severalaspects of the procedure may vary; said aspects include, but are notlimited to, the temporal regime of administering the therapeuticmodalities (e.g., prophylactic and/or therapeutic agents), whether suchtherapeutic modalities are administered separately or as an admixture,and the frequency of administration of the therapeutic modalities.

Animal models for cancer can be used to assess the efficacy of anantibody, antibody conjugate, a combination therapy or pharmaceuticalcomposition of the invention. Examples of animal models for lung cancerinclude, but are not limited to, lung cancer animal models described byZhang & Roth (1994, In Vivo 8(5):755-69) and a transgenic mouse modelwith disrupted p53 function (see, e.g., Morris et al. J. La. State Med.Soc. 1998, 150(4):179-85). An example of an animal model for breastcancer includes, but is not limited to, a transgenic mouse thatoverexpresses cyclin D1 (see, e.g., Hosokawa et al., Transgenic Res.2001, 10(5), 471-8. An example of an animal model for colon cancerincludes, but is not limited to, a TCR b and p53 double knockout mouse(see, e.g., Kado et al., Cancer Res. 2001, 61(6):2395-8). Examples ofanimal models for pancreatic cancer include, but are not limited to, ametastatic model of PancO2 murine pancreatic adenocarcinoma (see, e.g.,Wang et al., Int. J. Pancreatol. 2001, 29(1):37-46) and nu-nu micegenerated in subcutaneous pancreatic tumors (see, e.g., Ghaneh et al.,Gene Ther. 2001, 8(3):199-208). Examples of animal models fornon-Hodgkin's lymphoma include, but are not limited to, a severecombined immunodeficiency (“SCID”) mouse (see, e.g., Bryant et al., LabInvest. 2000, 80(4), 553-73) and an IgHmu-HOX11 transgenic mouse (see,e.g., Hough et al., Proc. Natl. Acad. Sci. USA 1998, 95(23), 13853-8. Anexample of an animal model for esophageal cancer includes, but is notlimited to, a mouse transgenic for the human papillomavirus type 16 E7oncogene (see, e.g., Herber et al., J. Virol. 1996, 70(3):1873-81).Examples of animal models for colorectal carcinomas include, but are notlimited to, Apc mouse models (see, e.g., Fodde & Smits, Trends Mol. Med.2001, 7(8):369-73 and Kuraguchi et al., Oncogene 2000, 19(50), 5755-63).

In some embodiments of the invention, the efficacy of the therapeuticregimen in reducing the quantity, number, amount or percentage of cancerstem cells in animals (including humans) undergoing treatment can beevaluated using in vivo techniques. In these embodiments, an imagingagent is used which binds to biological moieties on cancer stem cells,e.g., cancer stem cell surface antigens. For instance, a fluorescent tagor radionuclide is covalently attached to an antibody that binds to acancer stem cell surface antigen. The medical practitioner can infusethe labeled antibody into the patient either untreated or undergoingtreatment, and then the practitioner can place the patient into a totalbody scanner/developer which can detect the attached label (e.g.,fluorescent tag or radionuclide). The scanner/developer (e.g., CT or MRIscanner) records the presence and bodily location of the bound antibody.In this manner, the mapping and quantitation of tag (e.g. fluorescence,radioactivity) in patterns (i.e., different from patterns of normal stemcells within a tissue) within a tissue or tissues indicates thetreatment efficacy within the patient's body. For example, a largesignal (relative to a reference range or a prior treatment date) at aparticular location indicates the presence of cancer stem cells. If thissignal is increased relative to a prior treatment date it suggests aworsening of the disease and failure of therapy. Alternatively, a signaldecrease indicates that therapy is working.

Similarly, in some embodiments of the invention, the efficacy of thetherapeutic regimen in reducing the quantity, number, amount orpercentage of cancer cells in animals (including humans) undergoingtreatment can be evaluated using in vivo techniques. In one embodiment,the medical practitioner performs the imaging technique with labeledmolecule that binds the surface of a cancer cell, e.g., a cancer cellsurface antigen. In this manner, the mapping and quantitation of tag(e.g., fluorescence, radioactivity) in patterns within a tissue ortissues indicates the treatment efficacy within the body of the patientundergoing treatment.

In a specific embodiment, the amount of cancer stem cells is detected invivo in a subject according to a method comprising the steps of: (a)administering to the subject an effective amount of a labeled cancerstem cell marker binding agent that binds to a cell surface marker foundon the cancer stem cells, and (b) detecting the labeled agent in thesubject following a time interval sufficient to allow the labeled agentto concentrate at sites in the subject where the cancer stem cellsurface marker is expressed. In accordance with this embodiment, thecancer stem cell surface marker-binding agent is administered to thesubject according to any suitable method in the art, for example,parenterally (e.g. intravenously), or intraperitoneally. In accordancewith this embodiment, the effective amount of the agent is the amountwhich permits the detection of the agent in the subject. This amountwill vary according to the particular subject, the label used, and thedetection method employed. For example, it is understood in the art thatthe size of the subject and the imaging system used will determine theamount of labeled agent needed to detect the agent in a subject usingimaging. In the case of a radiolabeled agent for a human subject, theamount of labeled agent administered is measured in terms ofradioactivity, for example from about 5 to 20 millicuries of 99Tc. Thetime interval following the administration of the labeled agent which issufficient to allow the labeled agent to concentrate at sites in thesubject where the cancer stem cell surface marker is expressed will varydepending on several factors, for example, the type of label used, themode of administration, and the part of the subject's body that isimaged. In a particular embodiment, the time interval that is sufficientis 6 to 48 hours, 6 to 24 hours, or 6 to 12 hours. In another embodimentthe time interval is 5 to 20 days or 5 to 10 days. The presence of thelabeled cancer stem cell surface marker-binding agent can be detected inthe subject using imaging means known in the art. In general, theimaging means employed depend upon the type of label used. Skilledartisans will be able to determine the appropriate means for detecting aparticular label. Methods and devices that may be used include, but arenot limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),fluorescence, chemiluminescence, and sonography. In a specificembodiment, the cancer stem cell surface marker-binding agent is labeledwith a radioisotope and is detected in the patient using a radiationresponsive surgical instrument (Thurston et al., U.S. Pat. No.5,441,050). In another embodiment, the cancer stem cell surfacemarker-binding agent is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the cancer stem cell surfacemarker-binding agent is labeled with a positron emitting metal and isdetected in the patient using positron emission-tomography. In yetanother embodiment, the cancer stem cell surface marker-binding agent islabeled with a paramagnetic label and is detected in a patient usingmagnetic resonance imaging (MRI).

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of an antibody,antibody conjugate or pharmaceutical composition disclosed herein forcancer or one or more symptoms thereof

5.12.3 Assessing Toxicity

The toxicity and/or efficacy of antibodies, antibody conjugates,pharmaceutical compositions, and regimens of the invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Therapeutic regimens that exhibit large therapeutic indicesare preferred. While therapeutic regimens that exhibit toxic sideeffects may be used, care should be taken to design a delivery systemthat targets such agents to the site of affected tissue in order tominimize potential damage to uninfected cells and, thereby, reduce sideeffects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the therapies for use inhumans. The dosage of such agents lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity to normal tissues. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any therapy used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test antibody or test antibody conjugate thatachieves a half-maximal inhibition of symptoms) as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. Levels of antibodies or antibody conjugates inplasma may be measured, for example, by high performance liquidchromatography.

5.13 Articles of Manufacture

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. Thepharmaceutical product may contain, for example, a conjugate of theinvention in a unit dosage form in a first container, and in a secondcontainer, sterile water for injection. Alternatively, the unit dosageform may be a solid suitable for oral, transdermal, intranasal, ortopical delivery.

In a specific embodiment, the unit dosage form is suitable forintravenous, intramuscular, intranasal, oral, topical or subcutaneousdelivery. Thus, the invention encompasses solutions, preferably sterile,suitable for each delivery route.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisorder in question. In other words, the article of manufactureincludes instruction means indicating or suggesting a dosing regimenincluding, but not limited to, actual doses, monitoring procedures,cancer cell counts, cancer stem cell counts, and other monitoringinformation.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i. v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises an antibody conjugate of the invention, and wherein saidpackaging material includes instruction means which indicate that saidantibody conjugate can be used to prevent, manage, treat, and/orameliorate one or more symptoms associated with cancer, or one or moresymptoms thereof by administering specific doses and using specificdosing regimens as described herein.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i. v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises antibody of the invention, and wherein said packagingmaterial includes instruction means which indicate that said antibodycan be used to prevent, manage, treat, and/or ameliorate one or moresymptoms associated with cancer, or one or more symptoms thereof byadministering specific doses and using specific dosing regimens asdescribed herein.

In specific embodiments, the article of manufacture include labeledantibodies that selectively or bind to stem cells, and preferably, thatselectively or bind to cancer stem cells. As such, the article containsa method to adjust the dosages used in the therapeutic regimens, and tomonitor the efficacy of the therapeutic regimen.

5.14 Bone Marrow Purging

The present invention provides a method for purging bone marrow orperipheral blood prior to autologous stem cell transplant, comprisingcontacting ex vivo bone marrow or peripheral blood obtained from a humanwith a composition comprising an amount of an antibody conjugate of theinvention for a time sufficient to purge the bone marrow or peripheralblood of cells expressing an alpha subunit of the IL-3 receptor alphasubunit. In certain embodiments of the invention, the antibodies andantibody conjugates (including those described in Section 5.3 and 5.4,infra) can be used to purge the bone marrow or peripheral blood. In anaspect of this embodiment, the number of bone marrow or peripheral bloodcells expressing an alpha subunit of the IL-3 receptor alpha subunitafter contacting with a conjugate of the invention is decreased by atleast 75%, 80%, 90%, 95%, or by at least 99% relative to a negativecontrol.

The present invention provides a method for performing an autologousbone marrow or peripheral blood stem cell transplant, comprisingadministering to a human an amount of purged bone marrow or peripheralblood effective to reconstitute hematopoietic function in said human,wherein said purged bone marrow or peripheral blood is bone marrow orperipheral blood obtained from said human previously contacted with anamount of an antibody conjugate of the invention for a time sufficientto purge the bone marrow or peripheral blood of cells expressing analpha subunit of the IL-3 receptor alpha subunit. Further, the presentinvention provides a composition comprising purged bone marrow orperipheral blood, wherein said purged bone marrow or peripheral blood isbone marrow or peripheral blood obtained from a human and contacted exvivo with an amount of an antibody conjugate of the invention for a timesufficient to purge the bone marrow or peripheral blood of cellsexpressing an alpha subunit of the IL-3 receptor alpha subunit. In oneaspect, the composition can further comprise a pharmaceuticallyacceptable carrier.

6. EXAMPLE

The following example is illustrative, and should not be viewed aslimiting the scope of the present invention. Reasonable variations, suchas those that occur to a reasonable artisan, can be made herein withoutdeparting from the scope of the present invention

6.1 Materials and Methods

Cells

Hybridoma cells of the monoclonal antibodies 26292, 32701, and 32716were obtained from StemLine Therapeutics, Inc. (New York, N.Y.) andgrown in DMEM medium with 10% FBS. TF-1 was obtained from American TypeCulture Collection (ATCC) and grown in RPMI 1640 medium with 10% FBS and2 ng/ml GM-CSF. Molm-13 and Molm-14 were provided by Dr. Robert J.Arceci (Kimmel Comprehensive Cancer Center, Johns Hopkins University)and grown in RPMI 1640 medium with 10% FBS. ML-1 was obtained from theNational Cancer Institute and grown in IMDM with 10% FBS. U937 was grownin RPMI 1640 medium with 10% FBS.

Construction and Purification of scFv-PE38 Immunotoxin

Total cellular RNA was isolated from hybridoma cells using the QiagenRNeasy mini kit. V_(H) and V_(L) cDNAs of the monoclonal antibodies wereobtained by a RACE method using SMART RACE cDNA amplification kit(Clontech) as described in Pastan I, Beers R, Bera TK. Recombinantimmunotoxins in the treatment of cancer. Methods Mol Biol 2004;248:503-18. The prepared cDNAs were used as the template for PCRreactions for V_(H) and V_(L). The PCR products were cloned into thepCR4®-TOPO® vector using the TOPO TA cloning kit (Invitrogen). Fiveindependent clones for each chain were sequenced to exclude thepossibility of PCR error. The obtained sequences were confirmed again bycomparing sequences in GenBank. Then the V_(H) and V_(L) were assembledinto a single chain Fv (scFv) and fused to PE38. The expression andpurification of scFv-PE38 was conducted as described in Pastan et al,above. 26292(Fv)-PE38-KDEL was constructed by replacing the PE38 portionfrom the expression plasmid with PE38 containing KDEL at the carboxylterminus in the expression plasmid. See Kreitman R J, Margulies I,Stetler-Stevenson M, et al. Cytotoxic activity of disulfide-stabilizedrecombinant immunotoxin RFB4(dsFv)-PE38 (BL22) toward fresh malignantcells from patients with B-cell leukemias. Clin Cancer Res 2000;6:1476-87.

FACS Analysis

FACS analysis was performed to measure the binding of the monoclonalantibodies or immunotoxins to CD123 expressing cells. For screeningmonoclonal antibodies with good binding affinity, 2.5×10⁵ TF-1 cellswere incubated with 0.5 μg/ml of the anti-CD123 monoclonal antibodies orMOPC 21 isotype control IgG₁ (Sigma) in 200 μl of PBS containing 5% FBSand 0.1% sodium azide. After incubation for 1 hour at 4° C., the cellswere washed twice with the same buffer and incubated with R-PE labeledgoat F(ab′)₂ anti-mouse IgG (BioSource). After washing, the cells weresuspended in 0.5 ml of the buffer, and the fluorescence associated withthe live cells was measured using a FACSCalibur instrument (BDBiosciences).

For cell staining using immunotoxins, 2.5×10⁵ cells were incubated withdifferent concentrations of immunotoxins for 1 hour at 4° C., followedby a rabbit anti-PE38 polyclonal antibody. The PE38 was detected withR-PE-labeled goat F(ab′)₂ anti-rabbit IgG (BioSource). For sequentialbinding in competition assays, the first immunotoxin was incubated withTF-1 for 1 hour, followed by the second immunotoxin for an additionalhour.

To evaluate CD123 expression on various leukemia cell lines, 2.5×10⁵cells were incubated with 10 μg/ml of R-PE labeled 9F5 (BD Pharmingen)or R-PE labeled isotype control IgG₁ (Sigma) for 1 hour at 4° C.QuantiBRITE PE Beads (BD Biosciences) was used as a PE fluorescencestandard. See Pannu K K, Joe E T, Iyer S B. Performance evaluation ofQuantiBRITE phycoerythrin beads. Cytometry 2001; 45:250-8.

Cytotoxicity Assay

Cytotoxicity on cell lines was measured by cell viability assay. Cellswere seeded into 96 well plates at a concentration of 2×10⁴ cells/well.Serial dilutions of immunotoxins in 0.2% human serum albumin (HSA) wereadded to the cells, resulting in final concentrations ranging from 0.1to 1000 ng/ml in 150 pl. After incubation for 48 hours, 10 μl of WST-8(Dojindo Molecular Technologies) was added to each well, and theincubation was carried out for 4 hours at 37° C. The absorbance of thesample at 450 nm was measured with a reference wavelength of 650 nm.Cytotoxicity is defined by IC₅₀, 50% inhibition of cell viability, whichis midway between the level of viability in the absence of toxin andthat in the presence of 10 μg/ml of cycloheximide. All experiments wereperformed in triplicate. For stability assays, 200 ng/ml of26292(Fv)-PE38 in HSA was incubated at 4° C. or 37° C. for varying timesbefore evaluation in the cytotoxicity assay.

6.2 Results

Screening Antibodies with High Affinity CD123

Seven anti-CD123 monoclonal antibodies were analyzed by flow cytometryfor their binding to CD123 on the surface of TF-1, a CD123-expressingleukemia cell line. In these studies, a concentration of 0.5 μg/ml (˜3nM) was used, to distinguish between high and low binders. As shown inFIG. 6A, monoclonal antibodies 26292, 32701, and 32716 had the highestfluorescence intensity and were the best binders in the panel.

Total RNA from the hybridoma cells (26292, 32701, and 32716) wasprepared and used for cDNA amplification to clone the heavy and lightchain Fvs. After sequencing confirmation, the Fvs of the V_(H) and V_(L)were linked together and cloned into an expression vector to constructthe scFv-PE38 recombinant immunotoxin (V_(H)-linker-V_(L)-PE38) asdescribed in Pastan et al above. Each immunotoxin was expressed in E.coli and purified to near 95% homogeneity as indicated by SDS-PAGEanalysis (FIG. 6B). The yields ranged from 2%-5% based on the amount ofinclusion body protein used as the starting material.

Binding Ability, Cytotoxic Activity and Stability of Anti-CD123Immunotoxins

Purified immunotoxins were incubated with TF-1 to test their binding toCD123 by flow cytometry (FIG. 7A). SS1P, an immunotoxin targeting (seeHassan R, Bera T, Pastan I. Mesothelin: a new target for immunotherapy.Clin Cancer Res 2004; 10:3937-42), which is not expressed on the surfaceof TF-1, was used as a negative control. After subtracting thebackground SS1P value, the median fluorescence intensities (MFIs) of26292(Fv)-PE38, 32701(Fv)-PE38, and 32716(Fv)-PE38 were 39.7, 18.9, and33.5, respectively, indicating that 26292(Fv)-PE38, 32701(Fv)-PE38, and32716(Fv)-PE38 bind specifically to TF-1. The apparent affinities of26292(Fv)-PE38, 32701(Fv)-PE38, and 32716(Fv)-PE38 on TF-1 determined byflow cytometry were 3.5 nM, 8.2 nM and 1.4 nM, respectively, showingthat these immunotoxins can bind TF-1 with high affinities. Typical datawith immunotoxin 26292(Fv)-PE38 is shown in FIG. 7B.

Cytotoxicity of these immunotoxins was tested on TF-1 (FIG. 7C).26292(Fv)-PE38 had the best activity with an IC50=200 ng/ml. The averageIC₅₀s from nine independent assays was 185 ng/ml. 32701(Fv)-PE38 and32716(Fv)-PE38 had lower activity and did not reach an IC50 at 1000ng/ml, where a 25% decrease in cell viability was observed. Because26292(Fv)-PE38 and 32716(Fv)-PE38 had similar binding abilities forCD123 (FIG. 7A and affinity data), it was surprising to find that theircytotoxic activities were so different. The possibility that theseimmunotoxins bound to different epitopes on CD123, which affected theirability to kill cells, was tested: Competition assays, in which cellswere incubated with an excess of one immunotoxin followed by an excessof the second, were carried out. If two immunotoxins bound to anoverlapping epitope, the fluorescence signal from the combination wouldbe close to that of a single immunotoxin, whereas if two immunotoxinsbound to different epitopes, the fluorescence signal from thecombination would be close to the sum of two individual values.

The results of these experiments are shown in Table 3. When immunotoxins32701(Fv)-PE38 and 32716(Fv)-PE38 were combined sequentially, thefluorescent signal was close to that of the first immunotoxin added. Incontrast, when 26292(Fv)-PE38 was combined with either 32701(Fv)-PE38 or32716(Fv)-PE38, the fluorescence was close to the sum of the twoindividual immunotoxins. These data indicate that immunotoxins32701(Fv)-PE38 and 32716(Fv)-PE38 bind to an overlapping epitope onCD123, whereas 26292(Fv)-PE38 binds to a different epitope. Similarresults using monoclonal antibodies instead of immunotoxins wereobtained (data not shown).

To be useful in therapy, an immunotoxin must be stable for several hoursat 37° C. in the circulation. Several single chain immunotoxins havebeen found to be unstable at 37° C. and disulfide bonded variants havebeen produced to overcome this difficulty. See Reiter Y, Brinkmann U,Lee B, et al. Engineering antibody Fv fragments for cancer detection andtherapy: disulfide-stabilized Fv fragments. Nat Biotechnol 1996;14:1239-45. To assess the stability of the most active immunotoxin,26292(Fv)-PE38 was incubated at 37° C. or 4° C. for 1, 3, and 6 hours(FIG. 7D). After incubation at 37° C. for 6 hours, there was no changein the activity of 26292(Fv)-PE38, showing that this immunotoxin isstable at a physiological temperature.

TABLE 3 Competition binding assay MFI-1^(st) IT MFT² if two ITs MFI =1^(st) + 2^(nd) observed have if two ITs have 1^(st) IT¹ 2^(nd) IT(Mean + S.D.) competition no competition 26292 / 71.2 + 5.3 / / 32701 /17.2 + 5.3 / / 32716 / 50.6 + 0.7 / / 32701 32716 28.8 + 3.5 17.2 67.832701 26292 84.8 + 3.0 17.2 88.4 32716 32701 50.3 + 1.8 50.6 67.8 3271626292 95.4 + 2.0 50.6 121.8  ¹IT, immunotoxin ²MFI, median fluorescenceintensities

-   -   60 nM of immunotoxin were added to TF-1 either alone or        Sequentially by another immunotoxin as indicated. Data are from        Three independent assays.

CD123 Expression and Cytotoxicity of 26292 on Leukemia Cell Lines

It is well established that antigen density on the cell surface oftarget cells plays an important role in the sensitivity of cells toimmunotoxins. See Alexander R L, Kucera G L, Klein B, et al. In vitrointerleukin-3 binding to leukemia cells predicts cytotoxicity of adiphtheria toxin/IL-3 fusion protein. Bioconjug Chem 2000; 11:564-8;Alexander R L, Ramage J, Kucera G L, et al. High affinity IL-3 receptoralpha subunit expression on blasts from patients with acute myelogenousleukemia correlates with cytotoxicity of a diphtheria toxin/IL-3 fusionprotein. Leuk Res 2001; 25:875-81; and Kreitman R J, Margulies I,Stetler-Stevenson M, et al. Cytotoxic activity of disulfide-stabilizedrecombinant immunotoxin RFB4(dsFv)-PE38 (BL22) toward fresh malignantcells from patients with B-cell leukemias. Clin Cancer Res 2000;6:1476-87. Therefore, CD123 expression levels on several AML cell lineswas assessed by flow cytometry using QuantiBRITE standard PE beads (FIG.8). For TF-1, 6900 CD123 sites were detected per cell, 9900 on Molm-13,13900 on Molm-14, and 1900 on ML-1 were detected. No expression on U937was detected.

Because these cell lines all had low to moderate CD123 expression, amore active derivative of 26292(Fv)-PE38 was produced by replacing theREDLK sequence at its carboxyl terminus with KDEL. The mutation of REDLKto KDEL has been reported to improve the cytotoxicity of immunotoxinstoward a variety of antigens. See Kreitman R J, Margulies I,Stetler-Stevenson M, et al. Cytotoxic activity of disulfide-stabilizedrecombinant immunotoxin RFB4(dsFv)-PE38 (BL22) toward fresh malignantcells from patients with B-cell leukemias. Clin Cancer Res 2000;6:1476-87; Kreitman R J, Pastan I. Importance of the glutamate residueof KDEL in increasing the cytotoxicity of Pseudomonas exotoxinderivatives and for increased binding to the KDEL receptor. Biochem J1995; 307:29-37; and Seetharam S, Chaudhary V K, FitzGerald D, et al.Increased cytotoxic activity of Pseudomonas exotoxin and two chimerictoxins ending in KDEL. J Biol Chem 1991; 266:17376-81. The cytotoxicityof 26292(Fv)-PE38 and 26292(Fv)-PE38-KDEL was determined on these celllines (FIG. 9). HB21(Fv)-PE40, an immunotoxin targeting the transferrinreceptor (highly expressed on most cancer cells; see Batra J K,Fitzgerald D J, Chaudhary V K, et al. Single-chain immunotoxins directedat the human transferrin receptor containing Pseudomonas exotoxin A ordiphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv). Mol CellBiol 1991; 11:2200-5), was used as a positive control to be sure thecells were sensitive to PE-based immunotoxins. Results of these assaysare summarized in Table 4.

TABLE 4 Summary of CD123 expression on AML cell lines and cytotoxicityof 26292 immunotoxin IC₅₀ (ng/ml) 26292(Fv)- CD123 sites 26292(Fv)-PE38- HB21(Fv)- AML cell lines per cell PE38 KDEL PE40 TF-1 6,900 200 382.2 Molm-13 9,900 200 30 20 Molm-14 13,900 >1,000 200 60 ML-11,900 >1,000 1,000 5.2 U937 0 >1,000 1,000 0.9

For all of the cell lines expressing CD123, the activity of theimmunotoxin was increased using the mutant immunotoxin containing KDEL;in U937, with no immunotoxin. In two of three cell lines with moderateCD123 expression (TF-1 and Molm-13), the KDEL variant was more activewith IC₅₀s falling from 200 ng/ml to 38 and 30 ng/ml, respectively.Molm-14 was less sensitive to the immunotoxins against CD123. The ML-1cell line had very low CD123 expression and was resistant to theimmunotoxin targeting CD123.

This example has described the production of three IL3Rα-cytotoxinconjugates that target IL3Rα and their activities on IL3Rα-expressingcell lines.

7. Equivalents

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description and accompanying drawings using no more thanroutine experimentation. Such modifications and equivalents are intendedto fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

1.-83. (canceled)
 84. A nucleic acid that encodes a single chainfragment variable (scFv) region of an antibody that binds CD123, whereinsaid nucleic acid comprises nucleotide sequences that encode thecomplementarity determining regions (CDRs) of the variable heavy (VH)domain and the variable light (VL) domain of said antibody, and whereinsaid CDRs comprise the VH domain CDR1 presented in SEQ ID NO: 13, the VHdomain CDR2 presented in SEQ ID NO: 14, the VH domain CDR3 presented inSEQ ID NO: 15, the VL domain CDR1 presented in SEQ ID NO: 18, the VLdomain CDR2 presented in SEQ ID NO: 19, and the VL domain CDR3 presentedin SEQ ID NO:
 20. 85. The nucleic acid of claim 84, wherein saidnucleotide sequence that encodes the CDRs of the VH domain of saidantibody comprises SEQ ID NO:
 11. 86. The nucleic acid of claim 84,wherein said nucleotide sequence that encodes the CDRs of the VL domainof said antibody comprises SEQ ID NO:
 16. 87. The nucleic acid of claim84, wherein said nucleotide sequence that encodes the CDRs of the VHdomain of said antibody comprises SEQ ID NO: 11 and wherein saidnucleotide sequence that encodes the CDRs of the VL domain of saidantibody comprises SEQ ID NO:
 16. 88. The nucleic acid of claim 84,wherein said nucleic acid encodes a fusion protein, said fusion proteincomprising said anti-CD123 scFv antibody region and a second protein.89. An expression vector comprising the nucleic acid of claim
 88. 90.The expression vector of claim 89, wherein said expression vector is aviral vector.
 91. An isolated mammalian cell comprising the expressionvector of claim
 89. 92. An isolated mammalian cell engineered to expressthe nucleic acid of claim
 88. 93. The mammalian cell of claim 92,wherein said mammalian cell is a human cell.
 94. The nucleic acid ofclaim 88, wherein said second protein is Pseudomonas exotoxin A.
 95. Thenucleic acid of claim 88, wherein said second protein is abrin, ricin A,cholera toxin, diphtheria toxin, tumor necrosis factor, α-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, a thrombotic agent, an anti-angiogenicagent, angiostatin, endostatin, a lymphokine, interleukin-1,interleukin-2, interleukin-6, granulocyte macrophage colony stimulatingfactor, and granulocyte colony stimulating factor, or a growth factor.96. An expression vector comprising the nucleic acid of claim
 84. 97.The expression vector of claim 96, wherein said expression vector is aviral vector.
 98. An isolated mammalian cell comprising the expressionvector of claim
 96. 99. An isolated mammalian cell engineered to expressthe nucleic acid of claim
 84. 100. The mammalian cell of claim 99,wherein said mammalian cell is a human cell.
 101. A nucleic acid thatencodes an anti-CD123 single chain fragment variable (scFv) antibodyregion, wherein said nucleic acid comprises SEQ ID NO: 11 and SEQ ID NO:16.
 102. The nucleic acid of claim 101, wherein said nucleic acidencodes a fusion protein, said fusion protein comprising said anti-CD123scFv antibody region and a second protein.
 103. An expression vectorcomprising the nucleic acid of claim
 102. 104. The expression vector ofclaim 103, wherein said expression vector is a viral vector.
 105. Anisolated mammalian cell comprising the expression vector of claim 103.106. An isolated mammalian cell engineered to express the nucleic acidof claim
 102. 107. The mammalian cell of claim 106, wherein saidmammalian cell is a human cell.
 108. The nucleic acid of claim 102,wherein said second protein is Pseudomonas exotoxin A.
 109. The nucleicacid of claim 102, wherein said second protein is abrin, ricin A,cholera toxin, diphtheria toxin, tumor necrosis factor, α-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, a thrombotic agent, an anti-angiogenicagent, angiostatin, endostatin, a lymphokine, interleukin-1,interleukin-2, interleukin-6, granulocyte macrophage colony stimulatingfactor, and granulocyte colony stimulating factor, or a growth factor.110. An expression vector comprising the nucleic acid of claim
 101. 111.The expression vector of claim 110, wherein said expression vector is aviral vector.
 112. An isolated mammalian cell comprising the expressionvector of claim
 110. 113. An isolated mammalian cell engineered toexpress the nucleic acid of claim
 101. 114. The mammalian cell of claim113, wherein said mammalian cell is a human cell.
 115. A nucleic acidthat encodes the variable heavy (VH) domain of monoclonal antibody32703, wherein said VH domain comprises SEQ ID NO:
 12. 116. The nucleicacid of claim 115, wherein said nucleic acid comprises SEQ ID NO: 11.117. The nucleic acid of claim 116, wherein said nucleic acid furthercomprises a nucleic acid sequence that encodes the variable light (VL)domain of monoclonal antibody 32703, wherein said VL domain comprisesSEQ ID NO:
 17. 118. The nucleic acid of claim 117, wherein said nucleicacid sequence that encodes the VL domain of monoclonal antibody 32703comprises SEQ ID NO:
 16. 119. A nucleic acid that encodes the variablelight (VL) domain of monoclonal antibody 32703, wherein said VL domaincomprises SEQ ID NO:
 17. 120. The nucleic acid of claim 119, whereinsaid nucleic acid comprises SEQ ID NO: 16.